U.S. patent application number 16/067817 was filed with the patent office on 2019-01-10 for anti-pd-l1 antibodies and uses thereof.
The applicant listed for this patent is PHARMAEXPLORER LIMITED. Invention is credited to Qing DUAN, Hongzhuan GU, Shaoping HU, Hu LIU, Lile LIU, Haishan LUO, Zhengrong SHUAI, Xiaolan SUN, Tatchi Teddy YANG, Xinxiu YANG.
Application Number | 20190010233 16/067817 |
Document ID | / |
Family ID | 59273286 |
Filed Date | 2019-01-10 |
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United States Patent
Application |
20190010233 |
Kind Code |
A1 |
LIU; Lile ; et al. |
January 10, 2019 |
ANTI-PD-L1 ANTIBODIES AND USES THEREOF
Abstract
Provided herein are anti-PD-L1 antibodies and antigen-binding
fragments thereof. Also provided are nucleic acids encoding the
antibodies, compositions containing the antibodies, and methods of
producing the antibodies and using the antibodies for treating or
preventing diseases such as cancer, infectious diseases and
autoimmune diseases.
Inventors: |
LIU; Lile; (Shanghai,
CN) ; YANG; Xinxiu; (Shanghai, CN) ; LUO;
Haishan; (Shanghai, CN) ; SHUAI; Zhengrong;
(Shanghai, CN) ; LIU; Hu; (Shanghai, CN) ;
HU; Shaoping; (Shanghai, CN) ; SUN; Xiaolan;
(Shanghai, CN) ; GU; Hongzhuan; (Shanghai, CN)
; DUAN; Qing; (Shanghai, CN) ; YANG; Tatchi
Teddy; (Shanghai, CN) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
PHARMAEXPLORER LIMITED |
Road Town, Tortola |
|
VG |
|
|
Family ID: |
59273286 |
Appl. No.: |
16/067817 |
Filed: |
December 27, 2016 |
PCT Filed: |
December 27, 2016 |
PCT NO: |
PCT/CN2016/112428 |
371 Date: |
July 2, 2018 |
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/76 20130101;
C07K 2317/732 20130101; A61P 35/00 20180101; C07K 2317/33 20130101;
A61P 31/00 20180101; A61P 37/04 20180101; C07K 2317/92 20130101;
C07K 2317/565 20130101; A61P 37/06 20180101; C07K 2317/21 20130101;
C07K 2317/56 20130101; A61P 35/02 20180101; C07K 16/2827 20130101;
C07K 2317/94 20130101; C07K 2317/24 20130101; A61P 43/00 20180101;
C07K 2317/734 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28 |
Foreign Application Data
Date |
Code |
Application Number |
Jan 4, 2016 |
CN |
201610003723.6 |
Claims
1. An isolated monoclonal antibody or antigen-binding fragment
thereof comprising a light chain complementarity determining region
1 (LCDR1), a LCDR2, a LCDR3, a heavy chain complementarity
determining region 1 (HCDR1), a HCDR2, and a HCDR3, having the
polypeptide sequences of: (1) SEQ ID NOs: 30, 31, 32, 26, 27, and
28, respectively (2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4,
respectively; (3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12,
respectively; (4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20,
respectively; (5) SEQ ID NOs: 38, 39, 40, 34, 35, and 36,
respectively; (6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44,
respectively; or (7) SEQ ID NOs: 54, 55, 56, 50, 51, and 52,
respectively; wherein the antibody or antigen-binding fragment
thereof binds PD-L1.
2. The isolated monoclonal antibody or antigen-binding fragment of
claim 1, comprising a heavy chain variable region having a
polypeptide sequence at least 95% identical to SEQ ID NO: 25, 1, 9,
17, 33, 41 or 49, or a light chain variable region having a
polypeptide sequence at least 95% identical to SEQ ID NO: 29, 5,
13, 21, 37, 45 or 53.
3. The isolated monoclonal antibody or antigen-binding fragment of
claim 1, comprising: a. a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 25, and a light chain variable
region having the polypeptide sequence of SEQ ID NO: 29; b. a heavy
chain variable region having the polypeptide sequence of SEQ ID NO:
1, and a light chain variable region having the polypeptide
sequence of SEQ ID NO: 5; c. a heavy chain variable region having
the polypeptide sequence of SEQ ID NO: 9, and a light chain
variable region having the polypeptide sequence of SEQ ID NO: 13;
d. a heavy chain variable region having the polypeptide sequence of
SEQ ID NO: 17, and a light chain variable region having the
polypeptide sequence of SEQ ID NO: 21; e. a heavy chain variable
region having the polypeptide sequence of SEQ ID NO: 33, and a
light chain variable region having the polypeptide sequence of SEQ
ID NO: 37; f. a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 41, and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 45; or g. a heavy chain
variable region having the polypeptide sequence of SEQ ID NO: 49,
and a light chain variable region having the polypeptide sequence
of SEQ ID NO: 53.
4. The isolated monoclonal antibody or antigen-binding fragment of
claim 1, wherein the antibody or antigen-binding fragment thereof
is chimeric.
5. The isolated monoclonal antibody or antigen-binding fragment of
claim 1, wherein the antibody or antigen-binding fragment thereof
is human.
6. The isolated monoclonal antibody or antigen-binding fragment of
claim 4, comprising a human heavy chain IgG4 constant region having
a S228P mutation, and a human antibody light chain kappa constant
region.
7. An isolated nucleic acid encoding the monoclonal antibody or
antigen-binding fragment of claim 1.
8. A vector comprising the isolated nucleic acid of claim 7.
9. A host cell comprising the nucleic acid of claim 8.
10. A pharmaceutical composition, comprising the isolated
monoclonal antibody or antigen-binding fragment of claim 1 and a
pharmaceutically acceptable carrier.
11. A method of blocking binding of PD-L1 to PD-1 and/or B7.1, or
augmenting secretion of IFN-.gamma. and IL-2 in a subject in need
thereof, comprising administering to the subject the pharmaceutical
composition of claim 10.
12. A method of treating an infectious disease or a graft versus
host disease in a subject in need thereof, comprising administering
to the subject the pharmaceutical composition of claim 10.
13. A method of treating a tumor in a subject in need thereof,
comprising administering to the subject the pharmaceutical
composition of claim 10, wherein the tumor is selected from the
group consisting of a solid tumor, a hematologic cancer, bladder
cancer, brain cancer, breast cancer, colon cancer, gastric cancer,
glioma, head cancer, leukemia, liver cancer, lung cancer, lymphoma,
myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer,
renal cancer, salivary cancer, stomach cancer, thymic epithelial
cancer, and thyroid cancer.
14. A method of producing the monoclonal antibody or
antigen-binding fragment of claim 1, comprising culturing a cell
comprising a nucleic acid encoding the monoclonal antibody or
antigen-binding fragment under conditions to produce the monoclonal
antibody or antigen-binding fragment, and recovering the antibody
or antigen-binding fragment from the cell or cell culture.
15. A method of producing a pharmaceutical composition comprising
the monoclonal antibody or antigen-binding fragment of claim 1,
comprising combining the monoclonal antibody or antigen-binding
fragment with a pharmaceutically acceptable carrier to obtain the
pharmaceutical composition.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is entitled to priority pursuant to Chinese
Patent Application No. 201610003723.6, filed Jan. 4, 2016, the
disclosure of which is incorporated by reference herein in its
entirety.
REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY
[0002] This application contains a sequence listing, which is
submitted electronically via EFS-Web as an ASCII formatted sequence
listing with a file name "Sequence Listing File", creation date of
Dec. 12, 2016 and having a size of about 39.1 kB. The sequence
listing submitted via EFS-Web is part of the specification and is
herein incorporated by reference in its entirety.
FIELD OF THE INVENTION
[0003] The invention relates to monoclonal anti-PD-L1 antibodies,
nucleic acids and expression vectors encoding the antibodies,
recombinant cells containing the vectors, and compositions
comprising the antibodies. Methods of making the antibodies, and
methods of using the antibodies to treat diseases including cancer
and autoimmune diseases are also provided.
BACKGROUND OF THE INVENTION
[0004] Tumor cells are able to evade the immune system by "editing"
host immunity in the tumor microenvironment in a variety of ways.
One way in which tumors carry out this so-called "cancer immune
escape" is by upregulating the expression of immune checkpoint
proteins, which are key regulators of the immune system, thus
suppressing the immune response. One such immunosuppressive
co-signal is mediated by the PD-1 receptor and its ligand
PD-L1.
[0005] PD-1 (Programmed Cell Death Protein 1 or CD279) is a type I
transmembrane protein that is one of the major immune checkpoint
molecules (Blank et al., 2005, Cancer Immunotherapy, 54:307-314).
PD-1 is primarily expressed on activated T cells, and it interacts
with the ligands PD-L1 (B7-H1 or CD274) and PD-L2 (B7-DC or CD273)
to induce an inhibitory signal resulting in reduced T cell
proliferation, cytokine production, and cytotoxic activity (Freeman
et al., 2000, J. Exp. Med., 192:1027-34).
[0006] PD-L1 (Programmed Cell Death 1 Ligand) is a type I
transmembrane protein that comprises an extracellular Ig-V like
domain, an Ig-C like domain, a transmembrane domain and an
intracellular C-terminus domain. PD-L1 is expressed on many cell
types, including T-cells, B-cells, endothelial, epithelial, and
antigen presenting cells, on cells of lung, liver and heart
tissues, and on several types of tumor cells. In contrast, PD-L2 is
narrowly expressed on professional antigen presenting cells, such
as dendritic cells and macrophages. (Dong H, Zhu G, Tamada K, Chen
L. Nature Med. 1999; 5:1365-1369.)
[0007] The interaction between PD-1 and PD-L1 is critical for
modulating the immune response, and it is the predominant mechanism
by which PD-L1-expressing tumor cells escape from immune
surveillance (Zippelius et al., 2015, Cancer Immunol Res.,
3(3):236-44). Persistent expression of PD-1 by T cells is highly
indicative of an exhausted phenotype, noted by a decrease in
effector function. This phenotype has been observed in various
types of tumor-infiltrating lymphocytes (TILs) and linked to poor
prognosis and tumor recurrence (Wherry, 2011, Nat. Immunol.,
12:492-99; Sheng Yao 2013 Nat Rev Drug Discov. 2013 12(2):
130-146).
[0008] Blocking of the PD-1/PD-L1 interaction can activate the
immune system and enhance anti-tumor immune responses, and it has
been demonstrated that in multiple syngeneic mouse tumor models,
blockade of PD-L1 or its receptor PD-1 promotes antitumor activity
(Hirano et al., 2005, Cancer Res., 65:1089-96). Thus, the
interaction between PD-1 and PD-L1 is an attractive target for
cancer immunotherapy.
[0009] Additionally, in chronic viral infections such as HIV,
HIV-specific CD8+ T cell function is impaired, expression of
cytokines and effector molecules is reduced, and the proliferation
ability of the T cells is decreased. Studies have shown that PD-1
expression in HIV-specific CD8+ T cells in HIV-infected individuals
is highly upregulated, suggesting that blocking the PD-1/PD-L1
pathway provides a potential treatment for chronic viral infection
and AIDS treatment. (Bowers N L, Helton E S, Huijbregts R P H, et
al. PLoS Pathog. 2014 March; 10 (3): e1003993).
[0010] Thus, therapeutic agents that block the PD-1/PD-L1 pathway
can provide a new treatment approach for various cancers, viral
infections such as HIV infections, T-cell depletion-related
conditions, and other immune diseases.
[0011] PD-1 also interacts with PD-L2, which is expressed in the
lung and kidney, to provide a unique negative signal that prevents
autoimmune responses. Thus, use of an anti-PD-1 antibody to block
the PD-1/PD-L1 pathway, which could also disrupt the PD-1/PD-L2
interaction, could prevent the PD-1/PD-L2-based inhibition of
autoimmune responses. Therefore, use of an anti-PD-1 antibody could
potentially result in autoimmune related pneumonia or nephritis.
However, anti-PD-L1 antibodies do not affect the PD-1/PD-L2
interaction, and their use could therefore minimize immune-related
adverse events.
[0012] Cancer immunotherapy, a recent breakthrough in cancer
treatment, employs a patient's own immune system to attack tumor
cells. Inhibitors of immune checkpoint proteins have the potential
to treat a variety of tumor types, such as metastatic melanoma,
lung cancer, breast cancer, renal cell carcinoma, etc. Recent
studies using cancer immunotherapy approaches have shown promising
results, especially in the case of metastatic carcinomas (Weinstock
and McDermott, 2015, Ther Adv Urol., 7(6):365-77). In addition,
cancer immunotherapy has shown great potential in the treatment of
blood cancers, including Hodgkin's lymphoma, multiple myeloma, bone
marrow dysplasia syndrome, non-Hodgkin's lymphoma, etc. (Zou and
Chen L, 2008, Nat Rev Immunol., 8(6):467-77). Side effects caused
by immune checkpoint inhibitors are negligible, reversible and
manageable, and an effective immune checkpoint inhibitor may
substantially improve the overall survival of cancer patients.
Immune checkpoint inhibitors can be used in combination with
targeted therapy or conventional radiotherapy and chemotherapy, and
such combinatorial therapy may be effective in the treatment of
many types of cancer. Clinical trials of anti-PD-L1 monoclonal
antibodies have been initiated by Genentech/Roche, Pfizer/Merck,
Serono and MedImmune.
[0013] Humanized anti-PD-L1 mAb (Tecentriq.RTM.) from
Roche/Genentech, has been approved for treatment of locally
advanced or metastatic urothelial carcinoma and non-small cell lung
cancer (NSCLC). In a clinical study of patients having advanced or
metastatic urothelial carcinoma disease progression following
neoadjuvant or adjuvant platinum-containing therapy, TECENTRIQ.TM.
shrank tumors (objective response rate, ORR) in 22% of people.
However, more than 2% of patients experienced Grade 3-4 adverse
reactions, and three patients experienced either sepsis,
pneumonitis or intestinal obstruction, which led to death.
TECENTRIQ.TM. was discontinued for adverse reactions in 3.2% of the
patients.
[0014] Thus, despite the progress, there is a need in the art for
more effective therapeutics comprising anti-PD-L1 antibodies that
effectively inhibit the PD-1/PD-L1 signaling activity while causing
minimal adverse side effects in humans.
BRIEF SUMMARY OF THE INVENTION
[0015] The invention satisfies this need by providing monoclonal
antibodies that specifically bind PD-L1 with high affinity and
induce the secretion of IFN-.gamma. and IL-2 by immune cells, as
measured in both a mixed lymphocyte reaction and a T lymphocyte
stimulation assay. In particular, the fully human anti-PD-L1
antibodies of the invention have a comparable affinity to PD-L1 as
Atezolizumab (Tecentriq.RTM., Roche-Genentech), a humanized IgG1
anti-PD-L1 mAb. Additionally, the antibodies demonstrate comparable
characteristics to Atezolizumab, but are predicted to have fewer
immunogenic adverse side effects in humans than Atezolizumab due to
the fact that they are fully human, rather than simply humanized
mAbs.
[0016] In one general aspect, the invention relates to isolated
monoclonal antibodies or antigen-binding fragments thereof that
bind PD-L1.
[0017] According to a particular aspect, the invention relates to
an isolated monoclonal antibody or antigen-binding fragment thereof
comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the
polypeptide sequences of:
[0018] (1) SEQ ID NOs: 30, 31, 32, 26, 27, and 28,
respectively;
[0019] (2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
[0020] (3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12,
respectively;
[0021] (4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20,
respectively;
[0022] (5) SEQ ID NOs: 38, 39, 40, 34, 35, and 36,
respectively;
[0023] (6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
or
[0024] (7) SEQ ID NOs: 54, 55, 56, 50, 51, and 52,
respectively;
wherein the antibody or antigen-binding fragment thereof binds
PD-L1, preferably binds specifically to human PD-L1.
[0025] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fragment thereof, comprising a heavy chain variable region having a
polypeptide sequence at least 80%, preferably at least 85% or 90%,
or more preferably at least 95%, identical to SEQ ID NO: 25, 1, 9,
17, 33, 41 or 49, or a light chain variable region having a
polypeptide sequence at least 80%, preferably at least 85% or 90%,
or more preferably at least 95%, identical to SEQ ID NO: 29, 5, 13,
21, 37, 45 or 53.
[0026] According to one embodiment, the isolated monoclonal
antibody or antigen-binding fragment thereof of the invention is a
human/rat chimeric.
[0027] According to another embodiment, the isolated monoclonal
antibody or antigen-binding fragment thereof of the invention is
human.
[0028] According to yet another embodiment, the isolated monoclonal
antibody or antigen-binding fragment thereof of the invention
further comprises a constant region, preferably a human heavy chain
IgG4 constant region, more preferably a human heavy chain IgG4
constant region having one or more mutations, such as a S228P
mutation, and a human antibody light chain kappa constant
region.
[0029] In another general aspect, the invention relates to an
isolated nucleic acid encoding a monoclonal antibody or
antigen-binding fragment thereof of the invention.
[0030] In another general aspect, the invention relates to a vector
comprising an isolated nucleic acid encoding a monoclonal antibody
or antigen-binding fragment thereof of the invention.
[0031] In another general aspect, the invention relates to a host
cell comprising an isolated nucleic acid encoding a monoclonal
antibody or antigen-binding fragment thereof of the invention.
[0032] In another general aspect, the invention relates to a
pharmaceutical composition, comprising an isolated monoclonal
antibody or antigen-binding fragment thereof of the invention and a
pharmaceutically acceptable carrier.
[0033] In another general aspect, the invention relates to a method
of blocking the binding of PD-L1 to PD-1 and/or to B7.1, or a
method of augmenting secretion of IFN-.gamma. and IL-2, in a
subject in need thereof, comprising administering to the subject a
pharmaceutical composition of the invention.
[0034] In another general aspect, the invention relates to a method
of treating a disease, disorder or condition, preferably an
infectious disease, an inflammatory disease, an immune disease, an
autoimmune disease, or a graft versus host disease in a subject in
need thereof, comprising administering to the subject a
pharmaceutical composition of the invention.
[0035] In another general aspect, the invention relates to a method
of treating a hyperproliferative disease in a subject in need
thereof, comprising administering to the subject a pharmaceutical
composition of the invention. The hyperproliferative disease can be
a non-malignant disease, including but not limited to,
atherosclerosis, benign hyperplasia, and benign prostatic
hypertrophy. The hyperproliferative disease can also be a tumor, or
a malignant disease. The tumor can be selected from the group
consisting of a solid tumor, a hematologic cancer, bladder cancer,
biliary cancer, brain cancer, breast cancer, colon cancer,
esophageal cancer, gastric cancer, glioma, head cancer, leukemia,
liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian
cancer, melanoma, pancreatic cancer, renal cancer, salivary cancer,
stomach cancer, thymic epithelial cancer, and thyroid cancer.
[0036] In another general aspect, the invention relates to a method
of producing a monoclonal antibody or antigen-binding fragment
thereof of the invention, comprising culturing a cell comprising a
nucleic acid encoding the monoclonal antibody or antigen-binding
fragment under conditions to produce the monoclonal antibody or
antigen-binding fragment thereof, and recovering the antibody or
antigen-binding fragment thereof from the cell or cell culture.
[0037] In another general aspect, the invention relates to a method
of producing a pharmaceutical composition comprising a monoclonal
antibody or antigen-binding fragment thereof of the invention,
comprising combining the monoclonal antibody or antigen-binding
fragment thereof with a pharmaceutically acceptable carrier to
obtain the pharmaceutical composition.
[0038] Other aspects, features and advantages of the invention will
be apparent from the following disclosure, including the detailed
description of the invention and its preferred embodiments and the
appended claims.
BRIEF DESCRIPTION OF THE DRAWINGS
[0039] The foregoing summary, as well as the following detailed
description of the invention, will be better understood when read
in conjunction with the appended drawings. It should be understood
that the invention is not limited to the precise embodiments shown
in the drawings.
[0040] In the drawings:
[0041] FIG. 1 shows the binding activity of biotin-labeled
PD-L1-hFc protein to its receptor PD-1-hFc;
[0042] FIG. 2 shows the flow cytometry profile of HEK293 cells
stably transfected with PD-L1 protein;
[0043] FIG. 3 shows the ELISA measurement of serum antibody titers
of H2L2 transgenic mice after immunization with PD-L1 protein,
where 1681-1685 represent the mouse ID numbers;
[0044] FIG. 4A and FIG. 4B show the binding activity of chimeric
anti-PD-L1 antibodies according to embodiments of the invention to
human PD-L1-hFc protein, as measured by ELISA;
[0045] FIG. 5 shows the binding activity of chimeric anti-PD-L1
antibodies according to embodiments of the invention to cyno monkey
PD-L1-hFc protein, as measured by ELISA;
[0046] FIG. 6 shows the binding activity of chimeric anti-PD-L1
antibodies according to embodiments of the invention to PD-L1 or
PD-L2, as measured by ELISA;
[0047] FIG. 7A and FIG. 7B show the cell-based binding activity of
chimeric anti-PD-L1 antibodies according to embodiments of the
invention to CHO-K1-hPD-L1, as measured by flow cytometry;
[0048] FIG. 8A and FIG. 8B show the cell-based binding activity of
chimeric anti-PD-L1 antibodies according to embodiments of the
invention to CHO-K1-cPD-L1, as measured by flow cytometry;
[0049] FIG. 9A and FIG. 9B show the inhibition of binding of PD-L1
protein to its receptor PD-1 by chimeric anti-PD-L1 antibodies
according to embodiments of the invention, as measured by a
protein-based receptor ligand blocking assay;
[0050] FIG. 10A and FIG. 10B show the inhibition of binding of
PD-L1 protein to its ligand B7.1 by chimeric anti-PD-L1 antibodies
according to embodiments of the invention, as measured by a
protein-based receptor ligand blocking assay;
[0051] FIG. 11 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a T cell stimulation assay using PBMC's;
[0052] FIG. 12 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a T cell stimulation assay using PBMC's;
[0053] FIG. 13 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction using PBMC's from donor 1;
[0054] FIG. 14 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction assay using PBMC's from donor 1;
[0055] FIG. 15 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 2;
[0056] FIG. 16 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 3;
[0057] FIG. 17 shows the effect of chimeric anti-PD-L1 antibody
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 4;
[0058] FIG. 18 shows the effect of chimeric anti-PD-L1 antibody
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 5;
[0059] FIG. 19 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 6;
[0060] FIG. 20 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 7;
[0061] FIG. 21 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction assay using PBMC's from donor 6;
[0062] FIG. 22 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction assay using PBMC's from donor 7;
[0063] FIG. 23 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 8;
[0064] FIG. 24 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IFN-.gamma. secretion
in a mixed lymphocyte reaction assay using PBMC's from donor 9;
[0065] FIG. 25 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction assay using PBMC's from donor 8;
[0066] FIG. 26 shows the effect of chimeric anti-PD-L1 antibodies
according to embodiments of the invention on IL-2 secretion in a
mixed lymphocyte reaction assay using PBMC's from donor 9;
[0067] FIG. 27A and FIG. 27B show the binding to human PD-L1
protein by fully human anti-PD-L1 antibodies according to
embodiments of the invention, as measured by ELISA;
[0068] FIG. 28A and FIG. 28B show the binding to cyno PD-L1 protein
by fully human anti-PD-L1 antibodies according to embodiments of
the invention, as measured by ELISA;
[0069] FIG. 29 shows the cell-based binding activity of fully human
anti-PD-L1 antibodies according to embodiments of the invention to
CHO-K1-hPD-L1, as measured by flow cytometry;
[0070] FIG. 30 shows the cell-based binding activity of fully human
anti-PD-L1 antibodies according to embodiments of the invention to
CHO-K1-cPD-L1, as measured by flow cytometry;
[0071] FIG. 31A and FIG. 31B show the inhibition of binding of
PD-L1 protein to its receptor PD-1 by fully human anti-PD-L1
antibodies according to embodiments of the invention, as measured
by a protein-based receptor ligand blocking assay;
[0072] FIG. 32A and FIG. 32B show the inhibition of binding of
PD-L1 protein to its ligand B7.1 by fully human anti-PD-L1
antibodies according to embodiments of the invention, as measured
by a protein-based receptor ligand blocking assay;
[0073] FIG. 33 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0074] FIG. 34 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0075] FIG. 35 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0076] FIG. 36 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0077] FIG. 37 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0078] FIG. 38 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IL-2
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0079] FIG. 39 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0080] FIG. 40 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0081] FIG. 41 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0082] FIG. 42 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a mixed lymphocyte reaction assay using PBMC's;
[0083] FIG. 43 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a T cell stimulation assay using PBMC's;
[0084] FIG. 44 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a T cell stimulation assay using PBMC's;
[0085] FIG. 45 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a T cell stimulation assay using PBMC's;
[0086] FIG. 46 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a T cell stimulation assay using PBMC's;
[0087] FIG. 47 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on IFN-.gamma.
secretion in a T cell stimulation assay using PBMC's;
[0088] FIG. 48 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on
Antibody-Dependent Cellular Cytotoxicity (ADCC);
[0089] FIG. 49 shows the effect of fully human anti-PD-L1
antibodies according to embodiments of the invention on
Complement-Dependent Cytotoxicity;
[0090] FIG. 50 shows the thermostability of fully human anti-PD-L1
antibodies according to embodiments of the invention, as measured
by Differential Scanning Calorimetry (DSC);
[0091] FIG. 51 shows the freeze/thaw stability of fully human
anti-PD-L1 antibodies according to embodiments of the invention;
and
[0092] FIG. 52 shows the solubility of fully human anti-PD-L1
antibodies according to embodiments of the invention.
DETAILED DESCRIPTION OF THE INVENTION
[0093] Various publications, articles and patents are cited or
described in the background and throughout the specification; each
of these references is herein incorporated by reference in its
entirety. Discussion of documents, acts, materials, devices,
articles or the like which has been included in the present
specification is for the purpose of providing context for the
invention. Such discussion is not an admission that any or all of
these matters form part of the prior art with respect to any
inventions disclosed or claimed.
[0094] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning commonly understood to one of
ordinary skill in the art to which this invention pertains.
Otherwise, certain terms used herein have the meanings as set in
the specification. All patents, published patent applications and
publications cited herein are incorporated by reference as if set
forth fully herein. It must be noted that as used herein and in the
appended claims, the singular forms "a," "an," and "the" include
plural reference unless the context clearly dictates otherwise.
[0095] Unless otherwise stated, any numerical value, such as a
concentration or a concentration range described herein, are to be
understood as being modified in all instances by the term "about."
Thus, a numerical value typically includes .+-.10% of the recited
value. For example, a concentration of 1 mg/mL includes 0.9 mg/mL
to 1.1 mg/mL. Likewise, a concentration range of 1% to 10% (w/v)
includes 0.9% (w/v) to 11% (w/v). As used herein, the use of a
numerical range expressly includes all possible subranges, all
individual numerical values within that range, including integers
within such ranges and fractions of the values unless the context
clearly indicates otherwise.
[0096] The invention generally relates to isolated anti-PD-L1
antibodies, nucleic acids and expression vectors encoding the
antibodies, recombinant cells containing the vectors, and
compositions comprising the antibodies. Methods of making the
antibodies, and methods of using the antibodies to treat diseases
including cancer and autoimmune diseases are also provided. The
antibodies of the invention possess one or more desirable
functional properties, including but not limited to high-affinity
binding to PD-L1, high specificity to PD-L1, the ability to block
the binding of PD-L1 to its binding partners, PD-1 and B7.1, and
the ability to stimulate secretion of the cytokines IFN-.gamma. and
IL-2.
[0097] In a general aspect, the invention relates to isolated
monoclonal antibodies or antigen-binding fragments thereof that
bind PD-L1.
[0098] As used herein, the term "antibody" is used in a broad sense
and includes immunoglobulin or antibody molecules including human,
humanized, composite and chimeric antibodies and antibody fragments
that are monoclonal or polyclonal. In general, antibodies are
proteins or peptide chains that exhibit binding specificity to a
specific antigen. Antibody structures are well known.
Immunoglobulins can be assigned to five major classes (i.e., IgA,
IgD, IgE, IgG and IgM), depending on the heavy chain constant
domain amino acid sequence. IgA and IgG are further sub-classified
as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4. Accordingly,
the antibodies of the invention can be of any of the five major
classes or corresponding sub-classes. Preferably, the antibodies of
the invention are IgG1, IgG2, IgG3 or IgG4. Antibody light chains
of vertebrate species can be assigned to one of two clearly
distinct types, namely kappa and lambda, based on the amino acid
sequences of their constant domains Accordingly, the antibodies of
the invention can contain a kappa or lambda light chain constant
domain According to particular embodiments, the antibodies of the
invention include heavy and/or light chain constant regions from
rat or human antibodies. In addition to the heavy and light
constant domains, antibodies contain an antigen-binding region that
is made up of a light chain variable region and a heavy chain
variable region, each of which contains three domains (i.e., CDR1,
CDR2, and CDR3). The light chain variable region domains are
alternatively referred to as LCDR1, LCDR2 and LCRD3, and the heavy
chain variable region domains are alternatively referred to as
HCDR1, HCRD2 and HCDR3.
[0099] As used herein, the term an "isolated antibody" refers to an
antibody which is substantially free of other antibodies having
different antigenic specificities (e.g., an isolated antibody that
specifically binds to PD-L1 is substantially free of antibodies
that do not bind to PD-L1). In addition, an isolated antibody is
substantially free of other cellular material and/or chemicals.
[0100] As used herein, the term "monoclonal antibody" refers to an
antibody obtained from a population of substantially homogeneous
antibodies, i.e., the individual antibodies comprising the
population are identical except for possible naturally occurring
mutations that may be present in minor amounts. The monoclonal
antibodies of the invention can be made by the hybridoma method,
phage display technology, single lymphocyte gene cloning
technology, or by recombinant DNA methods. For example, the
monoclonal antibodies can be produced by a hybridoma which includes
a B cell obtained from a transgenic nonhuman animal, such as a
transgenic mouse or rat, having a genome comprising a human heavy
chain transgene and a light chain transgene.
[0101] As used herein, the term "antigen-binding fragment" refers
to an antibody fragment such as, for example, a diabody, a Fab, a
Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment
(dsFv), a (dsFv).sub.2, a bispecific dsFv (dsFv-dsFv'), a disulfide
stabilized diabody (ds diabody), a single-chain antibody molecule
(scFv), a single domain antibody (sdab) an scFv dimer (bivalent
diabody), a multispecific antibody formed from a portion of an
antibody comprising one or more CDRs, a camelized single domain
antibody, a nanobody, a domain antibody, a bivalent domain
antibody, or any other antibody fragment that binds to an antigen
but does not comprise a complete antibody structure. An
antigen-binding fragment is capable of binding to the same antigen
to which the parent antibody or a parent antibody fragment binds.
According to particular embodiments, the antigen-binding fragment
comprises a light chain variable region, a light chain constant
region, and an Fd segment of the constant region of the heavy chain
According to other particular embodiments, the antigen-binding
fragment comprises Fab and F(ab').
[0102] As used herein, the term "single-chain antibody" refers to a
conventional single-chain antibody in the field, which comprises a
heavy chain variable region and a light chain variable region
connected by a short peptide of about 15 to about 20 amino acids.
As used herein, the term "single domain antibody" refers to a
conventional single domain antibody in the field, which comprises a
heavy chain variable region and a heavy chain constant region or
which comprises only a heavy chain variable region.
[0103] As used herein, the term "human antibody" refers to an
antibody produced by a human or an antibody having an amino acid
sequence corresponding to an antibody produced by a human made
using any technique known in the art. This definition of a human
antibody includes intact or full-length antibodies, fragments
thereof, and/or antibodies comprising at least one human heavy
and/or light chain polypeptide.
[0104] As used herein, the term "humanized antibody" refers to a
non-human antibody that is modified to increase the sequence
homology to that of a human antibody, such that the antigen-binding
properties of the antibody are retained, but its antigenicity in
the human body is reduced.
[0105] As used herein, the term "chimeric antibody" refers to an
antibody wherein the amino acid sequence of the immunoglobulin
molecule is derived from two or more species. The variable region
of both the light and heavy chains often corresponds to the
variable region of an antibody derived from one species of mammal
(e.g., mouse, rat, rabbit, etc.) having the desired specificity,
affinity, and capability, while the constant regions correspond to
the sequences of an antibody derived from another species of mammal
(e.g., human) to avoid eliciting an immune response in that
species.
[0106] As used herein, the term "PD-L1" refers to the Programmed
Cell Death 1 Ligand protein, a type I transmembrane receptor that
is expressed on many cell types, including T-cells, B-cells,
endothelial, epithelial, and antigen presenting cells, on cells of
lung, liver and heart tissues, and whose expression is highly
up-regulated on several types of tumor cells. The amino acid
sequence of a human PD-L1 is represented in GenBank Accession No.
NP_054862.1. Two binding partners for PD-L1 have been identified,
PD-1 and B7.1.
[0107] As used herein, an antibody that "specifically binds to
PD-L1" refers to an antibody that binds to PD-L1, preferably human
PD-L1, with a KD of 1.times.10.sup.-7M or less, preferably
1.times.10.sup.-8M or less, more preferably 1.times.10.sup.-9 M or
less, or 1.times.10.sup.-10 M or less. The term "KD" refers to the
dissociation constant, which is obtained from the ratio of Kd to Ka
(i.e., Kd/Ka) and is expressed as a molar concentration (M). KD
values for antibodies can be determined using methods in the art in
view of the present disclosure. For example, the KD of an antibody
can be determined by using surface plasmon resonance, such as by
using a biosensor system, e.g., a Biacore.RTM. system, or by using
bio-layer interferometry technology, such as a Octet RED96
system.
[0108] The smaller the value of the KD of an antibody, the higher
affinity that the antibody binds to a target antigen.
[0109] According to a particular aspect, the invention relates to
an isolated monoclonal antibody or antigen-binding fragment thereof
comprising LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the
polypeptide sequences of:
[0110] (1) SEQ ID NOs: 30, 31, 32, 26, 27, and 28,
respectively;
[0111] (2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
[0112] (3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12,
respectively;
[0113] (4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20,
respectively;
[0114] (5) SEQ ID NOs: 38, 39, 40, 34, 35, and 36,
respectively;
[0115] (6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
or
[0116] (7) SEQ ID NOs: 54, 55, 56, 50, 51, and 52,
respectively;
wherein the antibody or antigen-binding fragment thereof binds
PD-L1, preferably specifically binds to human PD-L1.
[0117] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fragment thereof of the invention, comprising a heavy chain
variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 25, 1, 9, 17, 33, 41 or 49, or a light
chain variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 29, 5, 13, 21, 37, 45 or 53. Preferably,
the isolated monoclonal antibody or antigen-binding fragment
thereof of the invention comprises a heavy chain variable region
having a polypeptide sequence at least 80%, preferably at least 85%
or 90%, or more preferably at least 95%, identical to SEQ ID NO:
25, 1, 9, 17, 33, 41 or 49, and a light chain variable region
having a polypeptide sequence at least 80%, preferably at least 85%
or 90%, or more preferably at least 95%, identical to SEQ ID NO:
29, 5, 13, 21, 37, 45 or 53, respectively.
[0118] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fragment thereof of the invention, comprising: [0119] a. a heavy
chain variable region having the polypeptide sequence of SEQ ID NO:
25, and a light chain variable region having the polypeptide
sequence of SEQ ID NO: 29; [0120] b. a heavy chain variable region
having the polypeptide sequence of SEQ ID NO: 1, and a light chain
variable region having the polypeptide sequence of SEQ ID NO: 5;
[0121] c. a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 9, and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 13; [0122] d. a heavy chain
variable region having the polypeptide sequence of SEQ ID NO: 17,
and a light chain variable region having the polypeptide sequence
of SEQ ID NO: 21; [0123] e. a heavy chain variable region having
the polypeptide sequence of SEQ ID NO: 33, and a light chain
variable region having the polypeptide sequence of SEQ ID NO: 37;
[0124] f. a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 41, and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 45; or [0125] g. a heavy
chain variable region having the polypeptide sequence of SEQ ID NO:
49, and a light chain variable region having the polypeptide
sequence of SEQ ID NO: 53.
[0126] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively. In
another embodiment, the isolated monoclonal antibody or
antigen-binding fragment thereof comprises a heavy chain variable
region having a polypeptide sequence at least 80%, preferably at
least 85% or 90%, or more preferably at least 95%, identical to SEQ
ID NO: 1, and a light chain variable region having a polypeptide
sequence at least 80%, preferably at least 85% or 90%, or more
preferably at least 95%, identical to SEQ ID NO: 5. Preferably, the
isolated monoclonal antibody or antigen-binding fragment thereof
comprises a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 1; and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 5.
[0127] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 14, 15, 16, 10, 11, and 12, respectively,
respectively. In another embodiment, the isolated monoclonal
antibody or antigen-binding fragment thereof comprises a heavy
chain variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 9, and a light chain variable region having
a polypeptide sequence at least 80%, preferably at least 85% or
90%, or more preferably at least 95%, identical to SEQ ID NO: 13.
Preferably, the isolated monoclonal antibody or antigen-binding
fragment thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 9; and a light chain variable
region having the polypeptide sequence of SEQ ID NO: 13.
[0128] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 22, 23, 24, 18, 19, and 20, respectively,
respectively. In another embodiment, the isolated monoclonal
antibody or antigen-binding fragment thereof comprises a heavy
chain variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 17, and a light chain variable region
having a polypeptide sequence at least 80%, preferably at least 85%
or 90%, or more preferably at least 95%, identical to SEQ ID NO:
21. Preferably, the isolated monoclonal antibody or antigen-binding
fragment thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 17; and a light chain variable
region having the polypeptide sequence of SEQ ID NO: 21.
[0129] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 30, 31, 32, 26, 27, and 28, respectively.
In another embodiment, the isolated monoclonal antibody or
antigen-binding fragment thereof comprises a heavy chain variable
region having a polypeptide sequence at least 80%, preferably at
least 85% or 90%, or more preferably at least 95%, identical to SEQ
ID NO: 25, and a light chain variable region having a polypeptide
sequence at least 80%, preferably at least 85% or 90%, or more
preferably at least 95%, identical to SEQ ID NO: 29. Preferably,
the isolated monoclonal antibody or antigen-binding fragment
thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 25; and a light chain variable
region having the polypeptide sequence of SEQ ID NO: 29.
[0130] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 38, 39, 40, 34, 35, and 36, respectively.
In another embodiment, the isolated monoclonal antibody or
antigen-binding fragment thereof comprises a heavy chain variable
region having a polypeptide sequence at least 80%, preferably at
least 85% or 90%, or more preferably at least 95%, identical to SEQ
ID NO: 33, and a light chain variable region having a polypeptide
sequence at least 80%, preferably at least 85% or 90%, or more
preferably at least 95%, identical to SEQ ID NO: 37. Preferably,
the isolated monoclonal antibody or antigen-binding fragment
thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 33; and a light chain variable
region having the polypeptide sequence of SEQ ID NO:37.
[0131] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively.
In another embodiment, the isolated monoclonal antibody or
antigen-binding fragment thereof comprises a heavy chain variable
region having a polypeptide sequence at least 80%, preferably at
least 85% or 90%, or more preferably at least 95%, identical to SEQ
ID NO: 41, and a light chain variable region having a polypeptide
sequence at least 80%, preferably at least 85% or 90%, or more
preferably at least 95%, identical to SEQ ID NO: 45. Preferably,
the isolated monoclonal antibody or antigen-binding fragment
thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 41; and a light chain variable
region having the polypeptide sequence of SEQ ID NO:45.
[0132] In one embodiment, the invention relates to an isolated
monoclonal antibody or antigen-binding fragment thereof, comprising
LCDR1, LCDR2, LCDR3, HCDR1, HCDR2 and HCDR3, having the polypeptide
sequences of SEQ ID NOs: 54, 55, 56, 50, 51, and 52, respectively.
In another embodiment, the isolated monoclonal antibody or
antigen-binding fragment thereof comprises a heavy chain variable
region having a polypeptide sequence at least 80%, preferably at
least 85% or 90%, or more preferably at least 95%, identical to SEQ
ID NO: 49, and a light chain variable region having a polypeptide
sequence at least 80%, preferably at least 85% or 90%, or more
preferably at least 95%, identical to SEQ ID NO: 53. Preferably,
the isolated monoclonal antibody or antigen-binding fragment
thereof comprises a heavy chain variable region having the
polypeptide sequence of SEQ ID NO: 49; and a light chain variable
region having the polypeptide sequence of SEQ ID NO:53.
[0133] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fmgment thereof of the invention, wherein the antibody or
antigen-binding fragment thereof is chimeric.
[0134] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fragment thereof of the invention, wherein the antibody or
antigen-binding fragment thereof is human.
[0135] According to another particular aspect, the invention
relates to an isolated monoclonal antibody or antigen-binding
fragment thereof of the invention, comprising a constant region,
preferably a human heavy chain IgG4 constant region, more
preferably a human heavy chain IgG4 constant region having a S228P
mutation (SEQ ID NO: 75), and a human antibody light chain constant
region, preferably a human light chain kappa constant region (SEQ
ID NO: 77).
[0136] In another general aspect, the invention relates to an
isolated nucleic acid encoding a monoclonal antibody or
antigen-binding fragment thereof of the invention. It will be
appreciated by those skilled in the art that the coding sequence of
a protein can be changed (e.g., replaced, deleted, inserted, etc.)
without changing the amino acid sequence of the protein.
Accordingly, it will be understood by those skilled in the art that
nucleic acid sequences encoding monoclonal antibodies or
antigen-binding fragments thereof of the invention can be altered
without changing the amino acid sequences of the proteins.
[0137] In another general aspect, the invention relates to a vector
comprising an isolated nucleic acid encoding a monoclonal antibody
or antigen-binding fragment thereof of the invention. Any vector
known to those skilled in the art in view of the present disclosure
can be used, such as a plasmid, a cosmid, a phage vector or a viral
vector. In some embodiments, the vector is a recombinant expression
vector such as a plasmid. The vector can include any element to
establish a conventional function of an expression vector, for
example, a promoter, ribosome binding element, terminator,
enhancer, selection marker, and origin of replication. The promoter
can be a constitutive, inducible or repressible promoter. A number
of expression vectors capable of delivering nucleic acids to a cell
are known in the art and can be used herein for production of an
antibody or antigen-binding fragment thereof in the cell.
Conventional cloning techniques or artificial gene synthesis can be
used to generate a recombinant expression vector according to
embodiments of the invention.
[0138] In another general aspect, the invention relates to a host
cell comprising an isolated nucleic acid encoding a monoclonal
antibody or antigen-binding fragment thereof of the invention. Any
host cell known to those skilled in the art in view of the present
disclosure can be used for recombinant expression of antibodies or
antigen-binding fragments thereof of the invention. In some
embodiments, the host cells are E. coli TG1 or BL21 cells (for
expression of, e.g., an scFv or Fab antibody) or CHO-K1 cells (for
expression of, e.g., a full-length IgG antibody). According to
particular embodiments, the recombinant expression vector is
transformed into host cells by conventional methods such as
chemical transfection, heat shock, or electroporation, where it is
stably integrated into the host cell genome such that the
recombinant nucleic acid is effectively expressed.
[0139] In another general aspect, the invention relates to a method
of producing a monoclonal antibody or antigen-binding fragment
thereof of the invention, comprising culturing a cell comprising a
nucleic acid encoding the monoclonal antibody or antigen-binding
fragment thereof under conditions to produce a monoclonal antibody
or antigen-binding fragment thereof of the invention, and
recovering the antibody or antigen-binding fragment thereof from
the cell or cell culture (e.g., from the supernatant). Expressed
antibodies or antigen-binding fragments thereof can be harvested
from the cells and purified according to conventional techniques
known in the art and as described herein.
[0140] In another general aspect, the invention relates to a
pharmaceutical composition, comprising an isolated monoclonal
antibody or antigen-binding fragment thereof of the invention and a
pharmaceutically acceptable carrier.
[0141] As used herein, the term "carrier" refers to any excipient,
diluent, filler, salt, buffer, stabilizer, solubilizer, oil, lipid,
lipid containing vesicle, microsphere, liposomal encapsulation, or
other material well known in the art for use in pharmaceutical
formulations. It will be understood that the characteristics of the
carrier, excipient or diluent will depend on the route of
administration for a particular application. As used herein, the
term "pharmaceutically acceptable carrier" refers to a non-toxic
material that does not interfere with the effectiveness of a
composition according to the invention or the biological activity
of a composition according to the invention. According to
particular embodiments, in view of the present disclosure, any
pharmaceutically acceptable carrier suitable for use in an antibody
pharmaceutical composition can be used in the invention.
[0142] In another general aspect, the invention relates to a method
of blocking the binding of PD-L1 to PD-1 or to B7.1, or of
augmenting secretion of IFN-.gamma. and IL-2 in a subject in need
thereof, comprising administering to the subject a pharmaceutical
composition of the invention.
[0143] The functional activity of antibodies and antigen-binding
fragments thereof that bind PD-L1 can be characterized by methods
known in the art and as described herein. Methods for
characterizing antibodies and antigen-binding fragments thereof
that bind PD-L1 include, but are not limited to, affinity and
specificity assays including Biacore, ELISA, and FACS analysis;
receptor ligand binding assays to detect blocking of the binding of
PD-L1 to PD-1 and/or B7.1; assays to detect induction of
lymphocytic cytokine production by the blocking of the binding of
PD-L1 to PD-1 and/or B7.1; cell cytotoxicity assays to detect the
presence of antibody-dependent cell-mediated cytotoxicity (ADCC),
and complement dependent cytotoxicity (CDC) activity of the
antibodies; experiments to detect the inhibition of tumor growth in
mouse tumor models; etc. According to particular embodiments, the
methods for characterizing antibodies and antigen-binding fragments
thereof that bind PD-L1 include those described in Examples 2-12
below.
[0144] In another general aspect, the invention relates to a method
of treating an infectious disease or a graft versus host disease in
a subject in need thereof, comprising administering to the subject
a pharmaceutical composition of the invention. In another general
aspect, the invention relates to a method of treating a tumor in a
subject in need thereof, comprising administering to the subject a
pharmaceutical composition of the invention.
[0145] As used herein, the term "subject" refers to an animal, and
preferably a mammal According to particular embodiments, the
subject is a mammal including a non-primate (e.g., a camel, donkey,
zebra, cow, pig, horse, goat, sheep, cat, dog, rat, rabbit, guinea
pig or mouse) or a primate (e.g., a monkey, chimpanzee, or human).
In particular embodiments, the subject is a human.
[0146] According to embodiments of the invention, the
pharmaceutical composition comprises a therapeutically effective
amount of the anti-PD-L1 antibody or antigen-binding fragment
thereof. As used herein, the term "therapeutically effective
amount" refers to an amount of an active ingredient or component
that elicits the desired biological or medicinal response in a
subject. A therapeutically effective amount can be determined
empirically and in a routine manner, in relation to the stated
purpose.
[0147] As used herein with reference to anti-PD-L1 antibodies or
antigen-binding fragments thereof, a therapeutically effective
amount means an amount of the anti-PD-L1 antibody or
antigen-binding fragment thereof that stimulates an immune response
in a subject in need thereof. Also as used herein with reference to
anti-PD-L1 antibodies or antigen-binding fragments thereof, a
therapeutically effective amount means an amount of the anti-PD-L1
antibody or antigen-binding fragment thereof that results in
treatment of a disease, disorder, or condition; prevents or slows
the progression of the disease, disorder, or condition; or reduces
or completely alleviates symptoms associated with the immune
disease, disorder, or condition.
[0148] According to particular embodiments, the disease, disorder
or condition to be treated is a hyperproliferative disease, an
infectious disease, an inflammatory disease, an immune disease, an
autoimmune disease, or a graft versus host disease. According to
more particular embodiments, the disease, disorder or condition to
be treated is an infectious disease, an inflammatory disease, an
immune disease, an autoimmune disease, or a graft versus host
disease. According to more particular embodiments, the disease,
disorder or condition to be treated is a non-malignant
hyperproliferative disease, including but not limited to,
atherosclerosis, benign hyperplasia, benign prostatic hypertrophy.
According to other particular embodiments, the disease, disorder or
condition to be treated is a tumor, or a malignant
hyperproliferative disease, preferably a tumor selected from the
group consisting of a solid tumor, a hematologic cancer, bladder
cancer, biliary cancer, brain cancer, breast cancer, colon cancer,
esophageal cancer, gastric cancer, glioma, head cancer, leukemia,
liver cancer, lung cancer, lymphoma, myeloma, neck cancer, ovarian
cancer, melanoma, pancreatic cancer, renal cancer, salivary cancer,
stomach cancer, thymic epithelial cancer, and thyroid cancer.
[0149] According to particular embodiments, a therapeutically
effective amount refers to the amount of therapy which is
sufficient to achieve one, two, three, four, or more of the
following effects: (i) reduce or ameliorate the severity of the
disease, disorder or condition to be treated or a symptom
associated therewith; (ii) reduce the duration of the disease,
disorder or condition to be treated, or a symptom associated
therewith; (iii) prevent the progression of the disease, disorder
or condition to be treated, or a symptom associated therewith; (iv)
cause regression of the disease, disorder or condition to be
treated, or a symptom associated therewith; (v) prevent the
development or onset of the disease, disorder or condition to be
treated, or a symptom associated therewith; (vi) prevent the
recurrence of the disease, disorder or condition to be treated, or
a symptom associated therewith; (vii) reduce hospitalization of a
subject having the disease, disorder or condition to be treated, or
a symptom associated therewith; (viii) reduce hospitalization
length of a subject having the disease, disorder or condition to be
treated, or a symptom associated therewith; (ix) increase the
survival of a subject with the disease, disorder or condition to be
treated, or a symptom associated therewith; (xi) inhibit or reduce
the disease, disorder or condition to be treated, or a symptom
associated therewith in a subject; and/or (xii) enhance or improve
the prophylactic or therapeutic effect(s) of another therapy.
[0150] The therapeutically effective amount or dosage can vary
according to various factors, such as the disease, disorder or
condition to be treated, the means of administration, the target
site, the physiological state of the subject (including, e.g., age,
body weight, health), whether the subject is a human or an animal,
other medications administered, and whether the treatment is
prophylactic or therapeutic. Treatment dosages are optimally
titrated to optimize safety and efficacy.
[0151] According to particular embodiments, the compositions
described herein are formulated to be suitable for the intended
route of administration to a subject. For example, the compositions
described herein can be formulated to be suitable for intravenous,
subcutaneous, or intramuscular administration.
[0152] As used herein, the terms "treat," "treating," and
"treatment" are all intended to refer to an amelioration or
reversal of at least one measurable physical parameter related to a
cancer, an inflammatory disease, disorder or condition, an immune
disease, disorder or condition, an autoimmune disease, disorder or
condition, or an infectious disease, disorder or condition, which
is not necessarily discernible in the subject, but can be
discernible in the subject. The terms "treat," "treating," and
"treatment," can also refer to causing regression, preventing the
progression, or at least slowing down the progression of the
disease, disorder, or condition. In a particular embodiment,
"treat," "treating," and "treatment" refer to an alleviation,
prevention of the development or onset, or reduction in the
duration of one or more symptoms associated with the disease,
disorder, or condition, such as a tumor or more preferably a
cancer. In a particular embodiment, "treat," "treating," and
"treatment" refer to prevention of the recurrence of the disease,
disorder, or condition. In a particular embodiment, "treat,"
"treating," and "treatment" refer to an increase in the survival of
a subject having the disease, disorder, or condition. In a
particular embodiment, "treat," "treating," and "treatment" refer
to elimination of the disease, disorder, or condition in the
subject.
[0153] According to particular embodiments, a composition used in
the treatment of a cancer, an inflammatory disease, disorder or
condition, an immune disease, disorder or condition, an autoimmune
disease, disorder or condition, or an infectious disease, disorder
or condition can be used in combination with another treatment
including, but not limited to, a chemotherapy, an anti-CD20 mAb, an
anti-CTLA-4 antibody, an antiangiogenic agent, a radiation therapy,
other immune-oncology drug, a targeted therapy, an anti-PD-L1
antibody or other anticancer drugs.
[0154] As used herein, the term "in combination," in the context of
the administration of two or more therapies to a subject, refers to
the use of more than one therapy. The use of the term "in
combination" does not restrict the order in which therapies are
administered to a subject. For example, a first therapy (e.g., a
composition described herein) can be administered prior to (e.g., 5
minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 4
hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72 hours,
96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, 8
weeks, or 12 weeks before), concomitantly with, or subsequent to
(e.g., 5 minutes, 15 minutes, 30 minutes, 45 minutes, 1 hour, 2
hours, 4 hours, 6 hours, 12 hours, 16 hours, 24 hours, 48 hours, 72
hours, 96 hours, 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6
weeks, 8 weeks, or 12 weeks after) the administration of a second
therapy to a subject.
[0155] In another general aspect, the invention relates to a method
of producing a pharmaceutical composition comprising a monoclonal
antibody or antigen-binding fragment thereof of the invention,
comprising combining a monoclonal antibody or antigen-binding
fragment thereof with a pharmaceutically acceptable carrier to
obtain the pharmaceutical composition.
Embodiments
[0156] The invention provides also the following non-limiting
embodiments.
[0157] Embodiment 1 is an isolated monoclonal antibody or
antigen-binding fragment thereof comprising LCDR1, LCDR2, LCDR3,
HCDR1, HCDR2 and HCDR3, having the polypeptide sequences of:
[0158] (1) SEQ ID NOs: 30, 31, 32, 26, 27, and 28,
respectively;
[0159] (2) SEQ ID NOs: 6, 7, 8, 2, 3, and 4, respectively;
[0160] (3) SEQ ID NOs: 14, 15, 16, 10, 11, and 12,
respectively;
[0161] (4) SEQ ID NOs: 22, 23, 24, 18, 19, and 20,
respectively;
[0162] (5) SEQ ID NOs: 38, 39, 40, 34, 35, and 36,
respectively;
[0163] (6) SEQ ID NOs: 46, 47, 48, 42, 43, and 44, respectively;
or
[0164] (7) SEQ ID NOs: 54, 55, 56, 50, 51, and 52,
respectively;
[0165] wherein the antibody or antigen-binding fragment thereof
binds PD-L1.
[0166] Embodiment 2 is the isolated monoclonal antibody or
antigen-binding fragment of Embodiment 1, comprising a heavy chain
variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 25, 1, 9, 17, 33, 41 or 49, or a light
chain variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 29, 5, 13, 21, 37, 45 or 53.
[0167] Embodiment 3 is the isolated monoclonal antibody or
antigen-binding fragment of Embodiment 2, comprising a heavy chain
variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 25, 1, 9, 17, 33, 41 or 49, and a light
chain variable region having a polypeptide sequence at least 80%,
preferably at least 85% or 90%, or more preferably at least 95%,
identical to SEQ ID NO: 29, 5, 13, 21, 37, 45 or 53,
respectively.
[0168] Embodiment 4 is the isolated monoclonal antibody or
antigen-binding fragment of Embodiment 3, comprising: [0169] (a) a
heavy chain variable region having the polypeptide sequence of SEQ
ID NO: 25, and a light chain variable region having the polypeptide
sequence of SEQ ID NO: 29; [0170] (b) a heavy chain variable region
having the polypeptide sequence of SEQ ID NO: 1, and a light chain
variable region having the polypeptide sequence of SEQ ID NO: 5;
[0171] (c) a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 9, and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 13; [0172] (d) a heavy chain
variable region having the polypeptide sequence of SEQ ID NO: 17,
and a light chain variable region having the polypeptide sequence
of SEQ ID NO: 21; [0173] (e) a heavy chain variable region having
the polypeptide sequence of SEQ ID NO: 33, and a light chain
variable region having the polypeptide sequence of SEQ ID NO: 37;
[0174] (f) a heavy chain variable region having the polypeptide
sequence of SEQ ID NO: 41, and a light chain variable region having
the polypeptide sequence of SEQ ID NO: 45; or [0175] (g) a heavy
chain variable region having the polypeptide sequence of SEQ ID NO:
49, and a light chain variable region having the polypeptide
sequence of SEQ ID NO: 53.
[0176] Embodiment 5 is the isolated monoclonal antibody or
antigen-binding fragment of any of Embodiments 1 to 4, wherein the
antibody or antigen-binding fragment thereof is chimeric.
[0177] Embodiment 6 is isolated monoclonal antibody or
antigen-binding fragment of any of Embodiments 1 to 4, wherein the
antibody or antigen-binding fragment thereof is human.
[0178] Embodiment 7 is the isolated antibody or antigen-binding
fragment of Embodiment 5 or 6, comprising a human heavy chain IgG4
constant region with a S228P mutation, and a human antibody light
chain kappa constant region.
[0179] Embodiment 8 is the isolated antibody or antigen-binding
fragment of any of Embodiments 1 to 7, wherein the antibody or
antigen-binding fragment binds to a human PD-L1 with a K.sub.D of
5.times.10.sup.-9 M or less, preferably a K.sub.D of
1.times.10.sup.-9 M or lessor 1.times.10.sup.-10 M or less, wherein
the K.sub.D is measured by surface plasmon resonance analysis, such
as by using a Biacore system, or by a bio-layer interferometry
technology, such as by using a Octet RED96 system.
[0180] Embodiment 9 is an isolated nucleic acid encoding the
monoclonal antibody or antigen-binding fragment of any of
Embodiments 1 to 8.
[0181] Embodiment 10 is a vector comprising the isolated nucleic
acid of Embodiment 9.
[0182] Embodiment 11 is a host cell comprising the nucleic acid of
Embodiment 10.
[0183] Embodiment 12 is a pharmaceutical composition, comprising
the isolated monoclonal antibody or antigen-binding fragment of any
of Embodiments 1 to 8and a pharmaceutically acceptable carrier.
[0184] Embodiment 13 is a method of blocking binding of PD-L1 to
PD-1 and/or B7.1, or augmenting secretion of IFN-.gamma. and IL-2
in a subject in need thereof, comprising administering to the
subject the pharmaceutical composition of Embodiment 12.
[0185] Embodiment 14 is a method of treating an infectious disease,
an inflammatory disease, an immune disease, an autoimmune disease,
or a graft versus host disease in a subject in need thereof,
comprising administering to the subject the pharmaceutical
composition of Embodiment 12.
[0186] Embodiment 15 is the method of Embodiment 14, further
comprising administering to the subject an additional agent for
treating the infectious disease, inflammatory disease, immune
disease, autoimmune disease, or graft versus host disease in the
subject in need thereof.
[0187] Embodiment 16 is a method of treating a hyperproliferative
disease in a subject in need thereof, comprising administering to
the subject the pharmaceutical composition of Embodiment 12,
[0188] Embodiment 17 is the method of Embodiment 16, wherein the
hyperproliferative disease is a non-malignant disease, preferably
selected from the group consisting of atherosclerosis, benign
hyperplasia and benign prostatic hypertrophy.
[0189] Embodiment 18 is the method of Embodiment 16, wherein the
hyperproliferative disease is a tumor, or a malignant disease,
preferably, the tumor is selected from the group consisting of a
solid tumor, a hematologic cancer, bladder cancer, biliary cancer,
brain cancer, breast cancer, colon cancer, esophageal cancer,
gastric cancer, glioma, head cancer, leukemia, liver cancer, lung
cancer, lymphoma, myeloma, neck cancer, ovarian cancer, melanoma,
pancreatic cancer, renal cancer, salivary cancer, stomach cancer,
thymic epithelial cancer, and thyroid cancer.
[0190] Embodiment 19 is the method of any of Embodiments 16-18,
further comprising administering to the subject an additional agent
for treating the hyperproliferative disease in the subject in need
thereof.
[0191] Embodiment 20 is a method of producing the monoclonal
antibody or antigen-binding fragment of any of Embodiments 1 to 8,
comprising culturing a cell comprising a nucleic acid encoding the
monoclonal antibody or antigen-binding fragment under conditions to
produce the monoclonal antibody or antigen-binding fragment, and
recovering the antibody or antigen-binding fragment from the cell
or cell culture.
[0192] Embodiment 21 is a method of producing a pharmaceutical
composition comprising the monoclonal antibody or antigen-binding
fragment of any of Embodiments 1 to 8, comprising combining the
monoclonal antibody or antigen-binding fragment with a
pharmaceutically acceptable carrier to obtain the pharmaceutical
composition.
[0193] Embodiment 22 is an isolated monoclonal antibody or
antigen-binding fragment of any of Embodiments 1 to 8 for use in
treating an infectious disease, an inflammatory disease, an immune
disease, an autoimmune disease, or a graft versus host disease in a
subject in need thereof.
[0194] Embodiment 23 is an isolated monoclonal antibody or
antigen-binding fragment of any of Embodiments 1 to 8 for use in
treating a hyperproliferative disease, such as a non-malignant
disease selected from the group consisting of atherosclerosis,
benign hyperplasia and benign prostatic hypertrophy, or a tumor
selected from the group consisting of a solid tumor, a hematologic
cancer, bladder cancer, biliary cancer, brain cancer, breast
cancer, colon cancer, esophageal cancer, gastric cancer, glioma,
head cancer, leukemia, liver cancer, lung cancer, lymphoma,
myeloma, neck cancer, ovarian cancer, melanoma, pancreatic cancer,
renal cancer, salivary cancer, stomach cancer, thymic epithelial
cancer, and thyroid cancer, in a subject in need thereof.
[0195] Embodiment 24 is an use of an isolated monoclonal antibody
or antigen-binding fragment of any of Embodiments 1 to 8 for
manufacturing a pharmaceutical composition in treating an
infectious disease, an inflammatory disease, an immune disease, an
autoimmune disease, or a graft versus host disease in a subject in
need thereof.
[0196] Embodiment 25 is an use of an isolated monoclonal antibody
or antigen-binding fragment of any of Embodiments 1 to 8 for
manufacturing a pharmaceutical composition in treating a
hyperproliferative disease, such as a non-malignant disease
selected from the group consisting of atherosclerosis, benign
hyperplasia and benign prostatic hypertrophy, or a tumor selected
from the group consisting of a solid tumor, a hematologic cancer,
bladder cancer, biliary cancer, brain cancer, breast cancer, colon
cancer, esophageal cancer, gastric cancer, glioma, head cancer,
leukemia, liver cancer, lung cancer, lymphoma, myeloma, neck
cancer, ovarian cancer, melanoma, pancreatic cancer, renal cancer,
salivary cancer, stomach cancer, thymic epithelial cancer, and
thyroid cancer in a subject in need thereof.
EXAMPLES
[0197] The following examples of the invention are to further
illustrate the nature of the invention. It should be understood
that the following examples do not limit the invention and that the
scope of the invention is to be determined by the appended
claims
Example 1
Generation of Anti-PD-L1 Antibodies
[0198] Human PD-L1 protein was used as an immunogen to generate
anti-PD-L1 antibodies. The uses of human immunoglobulin transgenic
mouse technology for the development and preparation of fully human
antibodies was first described by Abgenix (xeno mouse and Medarex
(HuMab "mouse"); Lonberg et al., 1994, Nature 368: 856-859; Lonberg
and Huszar, 1995, Internal Rev. Immunol. 13:65-93; Harding and
Lonberg, 1995, Ann. N.Y. Acad. Sci. 764:536-546).
[0199] Antibodies with high affinity (K.sub.D<1*10.sup.-9M) to
PD-L1 were obtained by carrying out pilot antibody production,
purification and validation. The antibodies, which are specific for
PD-L1 and do not cross react with other immune checkpoints such as
PD-L2, are able to block the binding of PD-L1 to PD-1 and to B7.1.
The amino acid sequences of the heavy and light chain variable
regions of the generated anti-PD-L1 antibodies were determined
using standard molecular biology methods and are summarized in
Table 1.
TABLE-US-00001 TABLE 1 SEQ ID NOs of the amino acid sequences of
the heavy chain variable regions, HCDRs, light chain variable
regions and LCDRs of chimeric anti-PD-L1 antibodies of the
invention SEQ ID NOs Heavy Chain Light Chain variable variable
Clone ID region CDR1 CDR2 CDR3 region CDR1 CDR2 CDR3 78B2D7 1 2 3 4
5 6 7 8 78A11F6 9 10 11 12 13 14 15 16 105C5D7 17 18 19 20 21 22 23
24 73D3G2 25 26 27 28 29 30 31 32 73G11B3 33 34 35 36 37 38 39 40
127D2F3 41 42 43 44 45 46 47 48 192D9E7 49 50 51 52 53 54 55 56
[0200] The heavy chain and light chain variable regions of the
anti-PD-L1 antibodies listed in Table 1 are encoded by the nucleic
acid sequences summarized in Table 2.
TABLE-US-00002 TABLE 2 SEQ ID NOs of the nucleic acid sequences of
the heavy chain and light chain variable regions of chimeric
anti-PD-L1 antibodies of the invention SEQ ID NOs (nucleotides of
the sequence corresponding to CDRs) Clone ID Heavy Chain Light
Chain variable region 78B2D7 57 58 (CDR1: nt 76-105) (CDR1: nt
70-102) (CDR2: nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-327)
(CDR3: nt 265-291) 78A11F6 59 60 (CDR1: nt 76-105) (CDR1: nt
70-102) (CDR2: nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-330)
(CDR3: nt 265-291) 105C5D7 61 62 (CDR1: nt 76-105) (CDR1: nt
70-102) (CDR2: nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-324)
(CDR3: nt 265-291) 73D3G2 63 64 (CDR1: nt 76-105) (CDR1: nt 70-102)
(CDR2: nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-330) (CDR3: nt
265-288) 73G11B3 65 66 (CDR1: nt 76-105) (CDR1: nt 70-102) (CDR2:
nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-330) (CDR3: nt
265-291) 127D2F3 67 68 (CDR1: nt 76-105) (CDR1: nt 70-102) (CDR2:
nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-324) (CDR3: nt
265-291) 192D9E7 69 70 (CDR1: nt 76-105) (CDR1: nt 70-102) (CDR2:
nt 148-198) (CDR2: nt 148-168) (CDR3: nt 295-327) (CDR3: nt
265-291)
[0201] Fully human versions of the anti-PD-L1 antibodies were
generated. The fully human anti-PD-L1 antibodies bound to the
extracellular domain of human PD-L1 with high affinity
(K.sub.D<1*10.sup.-9M), and blocked the binding of PD-L1 to its
binding partners, PD-1 and B7.1. The anti-PD-L1 antibodies do not
exhibit non-specific binding to human PD-L2. The biological
activities of the anti-PD-L1 antibodies were evaluated by mixed
lymphocyte and T cell stimulation assays, in which they increased
secretion of the IFN-.gamma. and IL-2 cytokines. While not wishing
to be bound by theory, it is believed that the anti-PD-L1
antibodies can be used to suppress PD-1-mediated signaling pathways
that negatively-regulate immune responses, and to therefore enhance
tumor-specific immune responses, either as a monotherapy or in
combination with anti-PD-1 monoclonal antibodies or other
anticancer drugs, such as a cancer immunotherapy, particularly for
patients having PD-L1 positive tumors. The anti-PD-L1 antibodies
can be used for the treatment of cancers and autoimmune
diseases.
Example 2
Preparation of Anti-PD-L1 Antibodies
[0202] (Step 1) Preparation of Immunogen A, PD-L1.sup.ECD-hFc
Protein (Also Referred to as PD-L1-hFc Herein)
[0203] The coding sequence of the human PD-L1 extracellular domain
(PD-L1.sup.ECD.sub.; SEQ ID NOs: 71 and 78), corresponding to amino
acids Phe19-Thr239 of SEQ ID NO: 72, was cloned along with the
coding sequence of human IgG Fc fragment (hFc) into a pCpC vector
(Invitrogen, #V044-50) using standard molecular biology cloning
techniques (Sambrook and Russell, 1989, Molecular cloning: a
laboratory manual, New York: Cold Spring Harbor Laboratory Press,
2nd ed.). HEK293 cells (Invitrogen) were transiently transfected
using polyethylenimine (PEI, Polysciences) with the plasmid and
expanded in FreeStyle 293 expression medium (Invitrogen) at
37.degree. C. After 4 days of expansion, the culture medium was
collected and centrifuged to remove cell components. The culture
supernatant, which contained the recombinant PD-L1.sup.ECD-hFc, was
subjected to Protein A chromatography (Mabselect, GE Healthcare).
The ultraviolet (UV) absorption (A280 nm) was monitored with a UV
detector, and the samples were washed with PBS (pH 7.2) until the
UV A280 nm absorption returned to baseline levels, at which point
the PD-L1.sup.ECD-hFc fusion protein was eluted from the Protein A
affinity column with 0.1M glycine hydrochloride (pH 2.5). The
sample was dialyzed with PBS phosphate buffer (pH 7.2) at 4.degree.
C. overnight. After dialysis, the purified PD-L1 immunogen was
passed through a 0.22 micron sterile filter, aliquoted and stored
at -80.degree. C.
[0204] Recombinant human PD-1 extracellular domain (PD-1.sup.ECD)
(corresponding to amino acids Leu25-Glu167 of Uniprot database
protein Q15116) fused to hFc was prepared in the same way that the
PD-L1.sup.ECD-hFc recombinant protein was prepared above.
[0205] Purified PD-L1.sup.ECD-hFc (also referred to as PD-L1-hFc
herein) was biotinylated by mixing and incubating the protein with
biotin (Sigma S3295).
[0206] To characterize the PD-L1.sup.ECD-hFc immunogen, the
sample's protein concentration and purity were determined, and the
immunogen's molecular weight and biological activity were
determined.
[0207] The biological activity of the PD-L1.sup.ECD-hFc immunogen
was determined by ELISA. Recombinant PD-1.sup.ECD-hFc protein was
diluted in PBS to a concentration of 1 .mu.g/mL, and 100 .mu.L of
the diluted PD-1.sup.ECD-hFc protein sample were added per well to
ELISA microplates, which were incubated overnight at 4.degree. C.
to coat the plates with the recombinant protein. The plates were
then blocked with ELISA blocking solution (containing 1% BSA, pH
7.4 PBS buffer, w/v) at 37.degree. C. for two hours and then
incubated with serial dilutions of the biotinylated
PD-L1.sup.ECD-hFc protein or a control protein (biotinylated
non-PD-L1.sup.ECD-hFc) for 1 hour at 37.degree. C. Horseradish
peroxidase (HRP) conjugated streptavidin (Sigma B2438) was added,
and the plates were incubated at room temperature for 30 minutes.
100 .mu.L of tetramethylbenzidine (TMB) were added, and the plates
were incubated at room temperature for 15 minutes. 50 .mu.L of 1N
HCl were added to terminate the reaction, and the OD450 nm was
determined by an ELISA plate reader.
[0208] FIG. 1 and Table 3 show the concentration-dependent binding
of the biotinylated PD-L1.sup.ECD-hFc to purified PD-1.sup.ECD-hFc
fusion protein. Binding to PD-1.sup.ECD-hFc was observed with
biotinylated PD-L1.sup.ECD-hFc, but not with the control protein
that does not contain the sequence of PD-L1.sup.ECD.
TABLE-US-00003 TABLE 3 Binding activities of biotinylated
PD-L1.sup.ECD-hFc with its receptor PD-1.sup.ECD-hFc OD.sub.450 nm
Biotinylated PD-L1.sup.ECD-hFc (.mu.g/mL) Batches 1 0.2 0.04 0.008
0.0016 0.0003 0.0001 0.00001 PD-1-hFc 2.965 2.985 2.955 2.31 0.595
0.265 0.12 0.17 Batch 1 PD-1-hFc 2.905 3.005 3.065 2.295 0.95 0.43
0.25 0.335 Batch 2 Control 0.51 0.1 0.49 0.135 0.19 0.255 0.12 0.09
Protein
[0209] (Step 2) Preparation of Immunogen B, HEK293 Cells Over
Expressing hPD-L1
[0210] The nucleotide sequence encoding human PD-L1 (SEQ ID NO: 73)
was subcloned into a pIRES vector (Clontech), and the plasmid was
prepared. HEK293 and CHO-K1 cells (Invitrogen) were transiently
transfected with the plasmid using X-treme GENE HP DNA Transfection
Reagent (Roche, Cat #06 366 236 001), and transformants were
selected in DMEM culture media containing 0.5 g/mL antibiotics and
10% (w/w) fetal bovine serum (FBS) for 2 weeks. A limited dilution
into a 96-well culture plate was carried out, and the plate was
incubated at 37.degree. C. with 5% (v/v) CO.sub.2 for approximately
2 weeks. After selection, monoclones were expanded in 6-well
plates, and the expanded clones were screened by flow cytometry
using commercially available anti-PD-L1 antibodies (R&D
Systems). Clones exhibiting higher growth rates and higher
fluorescence intensity as measured by FACS were further expanded
and cryospreserved in liquid nitrogen.
[0211] FIG. 2 shows the flow cytometry profile of the HEK293 cells
stably transfected with the recombinant hPD-L1 protein. The
percentage of FACS-positive cells is shown in Table 4 as an
indication of the expression levels of hPD-L1 protein.
TABLE-US-00004 TABLE 4 hPD-L1 expression levels of recombinant
HEK293 clones, as determined by FACS Recombinant cell PD-L1 mAb IgG
Control No. clone ID Gated (%) MFI Gated (%) MFI 1 293F-hPD-L1 1A10
97.66 72.06 0.87 3.4 2 293F-hPD-L1 1B4 98.07 72.81 0.4 3.21 3
293F-hPD-L1 1D3 95.03 66.92 0.53 3.01 4 293F-hPD-L1 1D4 98.52
104.01 0.57 3.05 5 293F-hPD-L1 1D6 94.55 52.18 2.03 4.22 6
293F-hPD-L1 1E4 96.89 84.65 0.98 2.99 7 293F-hPD-L1 1E6 96.08 55.54
2.33 3.94 8 293F-hPD-L1 1E8 94.35 63.85 0.44 3.18 9 293F-hPD-L1 1F4
96.06 87.35 0.79 3.33 10 293F-hPD-L1 1F5 98.17 64.91 0.14 2.94 11
293F-hPD-L1 1F7 97.01 68.24 3.36 4.37 12 293F-hPD-L1 1F9 58.76
20.65 1.09 3.49 13 293F-hPD-L1 1G4 97.26 82.5 1.35 3.78
[0212] (Step 3) Preparation of Immunogen C, hPD-L1 Expressing
Construct
[0213] The coding sequence of full length human PD-L1 protein (SEQ
ID NO: 73) was subcloned into a pcDNA3.1 vector (Invitrogen), and
the resulting plasmid was coated onto a 1.0 um colloidal gold
bullet (Bio-RAD) for subsequent immunization using a Helios gene
gun (Bio-rad No. 165-2431), following the instructions of the
Helios gene gun data sheet.
[0214] (Step 4) Hybridoma Cell Fusion and Antibody Screening
[0215] Human Ig Fc does not interact with the mouse Fc receptor, so
immune responses triggered in mice by hFc-containing immunogens are
weak, resulting in low efficiency generation of monoclonal
antibodies. Harbour H2L2 transgenic mice were generated by
introducing the gene encoding the human immunoglobulin (Ig)
variable region and the gene encoding the rat Ig constant region
into the mouse genome, such that the mice contained a chimeric Ig
comprising hV-rC, while expression of the mouse Ig was disabled (WO
2010/070263 A1). Harbour H2L2 transgenic mice are able to produce
comparable immune responses and antibody titers to wild type mice
(e.g., Balb/c).
[0216] (part 4A) 6-8 week old Harbour H2L2 transgenic mice (Beijing
Weitong Lihua) were immunized with Immunogen A, and the mice were
kept under Specific Pathogen Free (SPF) conditions. In the first
immunization, 50 .mu.g of Immunogen A was injected into the
abdominal cavity of each mouse along with 0.25 mL Complete Freund's
Adjuvant (CFA). To enhance the immune response, 50 .mu.g of
Immunogen A was injected into the abdominal cavity of each mouse
along with 0.25 Incomplete Freund's Adjuvant (IFA) two weeks after
the first immunization, and subsequent boosts were administered 3
weeks apart. Blood samples were collected one week after
immunization. The antibody titer and specificity in serum were
determined by ELISA and FACS analysis, and the results are shown in
FIG. 3 and Table 5. Table 5 shows that serum from mice immunized
with PD-L1.sup.ECD-hFc exhibited different levels of binding to
Immunogen A. The highest serum dilution was about one million. The
blank control was 1% (w/w) BSA. The OD450 nm values shown in Table
5 are the serum titer values from 7 days after the third boost, as
determined by ELISA.
TABLE-US-00005 TABLE 5 Serum titers of Harbour H2L2 transgenic mice
immunized with hPD-L1.sup.ECD-hFc fusion protein, as determined by
ELISA OD.sub.450 nm Serum dilution factors Animal No. 1:100
1:10.sup.3 1:10.sup.4 1:10.sup.5 1:10.sup.6 1:10.sup.7 Blank
1681(TB3) 3.3324 3.0881 1.4936 0.9037 0.8361 0.835 0.86 1682(TB3)
3.3709 3.2329 2.1931 1.0003 0.9066 0.8184 0.8188 1683(TB3) 3.3552
3.2973 2.4815 1.0582 0.8574 0.8171 0.8845 1684(TB3) 3.3781 3.3031
2.9768 1.3902 0.9094 0.866 0.9304 1685(TB3) 3.3477 3.0496 1.4451
0.9969 0.9212 0.8983 0.9492
[0217] (part 4B) 6-8 week old Harbour H2L2 transgenic mice (Beijing
Weitong Lihua) were immunized with Immunogen B, and the mice were
kept under SPF conditions. HEK293 cells were stably transfected
with the pIRES plasmid encoding full length hPD-L1 (see Example 2,
step 2)) using X-treme gene HP DNA Transfection reagent (Roche, #06
366 236 001). Cells were cultured in T-75 culture flasks. When the
cells reached 90% confluence, the media was aspirated, and the
cells were washed twice with DMEM medium (Invitrogen) and treated
with non-enzymatic cell dissociation buffer (Invitrogen) at
37.degree. C. until the cells were detached from the culture flask.
The cells were collected and washed twice with DMEM medium, and
cell counts were determined and adjusted to 2.times.10.sup.7
cells/mL using PBS buffer (pH 7.2). Mice were injected with 0.5 mL
of cell suspension for each immunization Two weeks after the first
immunization, a boost was administered, and subsequent boosts were
administered 3 weeks apart. Blood samples were collected one week
after immunization The antibody titer and specificity in serum were
determined by flow cytometry. After the second boost, the serum
titer was over 1:1000, as determined by flow cytometry.
[0218] (part 4C) 6-8 week old Harbour H2L2 transgenic mice (Beijing
Weitong Lihua) were immunized with Immunogen C, and the mice were
kept under SPF conditions. All mice were immunized with the Helios
gene gun 4 times, with 4 shots per immunization. Each shot
contained 1 .mu.g cDNA. Two weeks after the first immunization, a
boost was administered, and subsequent boosts were administered 3
weeks apart. Blood samples were collected one week after
immunization, and the serum titer was determined by ELISA and FACS.
The serum titer after the second boost was 1:1000, as determined by
flow cytometry, and over 1:10,000, as determined by ELISA.
[0219] Usually serum titers from mice immunized with immunogens A-C
could reach 1:1000 as determined by FACS after the third boost.
[0220] Prior to the completion of steps 4A-C above, mice with
specific immune responses against hPD-L1 were selected for fusion
and were given a final boost by intraperitoneal injection of 100
.mu.g of purified PD-L1.sup.ECD-hFc (for mice immunized with
Immunogen A or Immunogen C) or HEK293 cells stably transfected with
hPD-L1 (for mice immunized with Immunogen B). Five days later, the
mice were sacrificed, and their splenocytes were collected.
NH.sub.4OH was added to the splenocyte samples to a final
concentration of 1% (w/w) to lyse red blood cells in the sample.
The samples were centrifuged at 1000 rpm and washed three times
with DMEM culture media. The viability of the splenocytes was
determined, and viable splenocytes were then fused with mouse
myeloma cells SP2/0 (ATCC) at a ratio of 5:1 by the high efficiency
electric fusion method (see Methods in Enzymology, Vol. 220).
[0221] Fused cells were re-suspended in DMEM media containing 20%
FBS and 1.times. hypoxanthine-aminopterin-thymidine (HAT) medium
(w/w), and the concentration was adjusted to 10.sup.5 cells/200
.mu.L. 200 .mu.L of fused cells were added to each well of 96-well
plate, which was incubated at 37.degree. C., 5% CO.sub.2. 14 days
after cell fusion, hybridoma supernatants were collected and
screened by ELISA and Acumen (microplate cell assay). Clones with
an OD450 nm greater than 1.0 by ELISA and an MFI value greater than
100 by Acumen were expanded in a 24-well plate containing DMEM with
10% (w/w) heat-inactivated FBS at 37.degree. C., 5% (v/v) CO.sub.2.
Supernatants were collected after 3 days of culturing. The antibody
isotypes were determined, and their ability to bind to recombinant
PD-L1.sup.ECD protein and PD-L1-expressing cells was determined by
ELISA and flow cytometry (see Example 3). A receptor ligand binding
assay was performed to determine the blocking activity of the
hybridoma supernatant (see Examples 4-5).
[0222] Based on the 24-well plate screening results, clones with
OD450 nm values greater than 1.0 by the ELISA binding assay, MFI
values greater than 50 by the FACS binding assay, and inhibition
rates greater than 60% by the receptor ligand binding assay were
selected and subcloned. Subcloning was carried out by limited
dilution in a 96-well plate with DMEM media containing 10% (v/v)
FBS at 37.degree. C. and 5% (v/v) CO.sub.2. After 10 days of
culturing, the supernatants were collected and preliminarily
screened by ELISA and Acumen. Positive clones were expanded in a
24-well plate and cultured for 3 days. Supernatants were collected.
ELISA and FACS binding assays were performed to determine the
binding activity, and receptor ligand binding assays were performed
to assess the biological activity. The selection criteria was as
follows: OD450 nm>1.0 by ELISA, MFI value >50 by FACS, and
inhibition rate >60% by receptor ligand binding assay. Clones
that met the selection criteria were expanded in DMEM media
containing 10% (w/w) FBS at 37.degree. C., 5% (v/v) CO.sub.2 and
frozen in liquid nitrogen so that the hybridoma cells could be used
for subsequent antibody production and purification.
[0223] (Step 5) Production and Purification of Lead Candidate
Antibodies
[0224] The antibody concentrations from the hybridoma cells were
low, about 1-10 .mu.g/mL, and there was a large variation in
antibody concentrations. In addition, the FBS and the components of
the culture medium can interfere with the analysis. Therefore, it
was necessary to perform small scale antibody production and
purification (1-5 mg).
[0225] Hybridoma cells from Example 2 (part 4) were cultured in
T-75 culture flasks using Hybridoma serum free medium (Invitrogen),
and passaged for 3 generations. Cells were transferred to 2 L
culture flasks when the hybridoma cells were in good condition. 500
mL of production media was added to each culture flask, and the
cell density was adjusted to 10.sup.5 cells/mL. The culture flasks
were placed onto a rotary incubator at 37.degree. C. with a
rotating speed of 3 cycles per minute. Hybridoma cells were
cultured for 14 days, after which the supernatant was collected and
the cells were removed. The supernatants were then filtered through
0.45 micron filtration. The culture supernatants were then ready
for purification or storage at -30.degree. C.
[0226] Monoclonal antibodies were purified by passing the hybridoma
culture supernatants through 2 mL. Protein G columns (GE
Healthcare). Protein G columns were first equilibrated with PBS
buffer (pH 7.2), and the hybridoma culture supernatants were then
applied to the equilibrated Protein G columns with a constant flow
rate of 3 mL/minute. The columns were then washed with 4 volumes of
PBS buffer. The chimeric anti-PD-L1 antibodies were then eluted
with elution buffer (0.1M acetate buffer, pH 2.5), and the UV
absorbance of the eluates were monitored using a UV detector (A280
UV absorption peak). 10% of 1.0M Tris-HCL buffer was added to the
eluates to neutralize the pH, and the samples were sterile-filtered
by passing them through 0.22 micron filters. Sterile-filtered
purified chimeric anti-PD-L1 antibodies were obtained.
[0227] The concentration of purified chimeric anti-PD-L1 antibodies
was determined by UV absorbance (A280/1.4), and the purity and
endotoxin level (Lonza kit) were measured. The results of the
analyses are shown in Table 6. The purified chimeric anti-PD-L1
antibodies had endotoxin concentrations less than 1.0 EU/mg.
TABLE-US-00006 TABLE 6 Quality Control analysis of Purified
Chimeric PD-L1 mAbs from Hybridoma Protein Concentration Endotoxin
Clone ID Purity (.mu.g/mL) (EU/.mu.g) 78B2D7 >90% 0.4 <0.1
78A11F6 >90% 0.23 <0.1 105C5D7 >90% 0.5 <0.1 73D3G2
>90% 0.55 0.94 73G11B3 >90% 0.33 2.84 127D2F3 >90% 0.29
3.04 192D9E7 >90% 0.82 1.24
Example 3
Characterization of Lead Candidate Antibodies
[0228] (Part A) Detection of the Binding of Anti-PD-L1 Antibodies
to Recombinant PD-L1.sup.ECD-hFc Protein by ELISA
[0229] The binding of purified anti-PD-L1 antibodies from Example 2
to recombinant human PD-L1.sup.ECD-hFc protein, cyno
PD-L1.sup.ECD-hFc protein and other PD-L1 family-related immune
checkpoint protein PD-L2 was analyzed by ELISA to determine the
specificity of the antibodies.
[0230] Purified Immunogen A (hPD-L1.sup.ECD-hFc protein) from
Example 2, cyno PD-L1.sup.ECD-hFc protein (prepared using the
method for preparing Immunogen A; see Example 2, step 1),
comprising the amino acid sequence of the extracellular domain of
cyno PD-L1, which corresponds to amino acids Phe19-Thr239 of
Uniprot database protein G7PSE7), and other immune checkpoint
proteins (PD-L2 and NC-Fc) (R&D Systems) were diluted to 1
.mu.g/mL in PBS. 100 .mu.L of the diluted recombinant proteins were
added to each well of 96-well plate. The plates were sealed with
plastic film and incubated at 4.degree. C. overnight. The plates
were washed twice with wash buffer (PBS+0.01% (v/v) Tween20), and
incubated with blocking buffer (PBS+0.01% (v/v) Tween20+1% (w/w)
BSA) at room temperature for 2 hours. The blocking buffer was
aspirated away, and 100 .mu.L of purified anti-PD-L1 antibodies
were added to each well and incubated for 2 hours at 37.degree. C.
The plates were washed three times with wash buffer (PBS+0.01%
(v/v) Tween20). HRP-conjugated secondary antibody (Sigma) was added
to each well and incubated for 2 hours at 37.degree. C. After the
plates were washed three times with wash buffer, 100 .mu.L TMB
substrate were added to each well, and the plates were incubated
for 30 minutes at room temperature. 100 .mu.L stop solution (0.1N
HCl) was added to each well to stop the reaction. The absorbance at
450 nm was measured with an ELISA plate reader (384plus SpectraMax,
Molecular Devices). The results are shown in FIGS. 4-6 and in
Tables 7-9. The IgG control was human IgG.
TABLE-US-00007 TABLE 7 Binding activities of chimeric anti-PD-L1
mAbs to human PD-L1.sup.ECD-hFc, as measured by ELISA OD.sub.450 nm
Antibody concentration (nM) Clone ID 30 10 3.333 1.111 0.370 0.123
0.041 Blank 78B2D7 1.54 1.56 1.41 1.38 1.16 0.67 0.36 0.17 78A11F6
1.95 1.72 1.73 1.86 1.39 0.84 0.37 0.18 105C5D7 1.53 1.56 1.50 1.32
1.19 0.67 0.34 0.15 73D3G2 1.81 2.09 2.03 1.81 1.29 0.72 0.36 0.19
IgG Control 0.12 0.15 0.11 0.16 0.12 0.11 0.11 0.13 OD.sub.450 nm
Antibody concentration (nM) Clone ID 100 10 1 0.1 0.01 0.001 0.0001
0 73G11B3 2.69 2.73 2.70 0.88 0.28 0.21 0.20 0.19 127D2F3 2.63 2.70
2.80 1.65 0.38 0.24 0.20 0.21 192D9E7 2.70 2.69 2.77 1.55 0.40 0.25
0.21 0.20 IgG Control 2.69 2.73 2.70 0.88 0.28 0.21 0.20 0.19
TABLE-US-00008 TABLE 8 Binding activities of chimeric anti-PD-L1
mAbs to Cyno Monkey PD-L1.sup.ECD-hFc, as measured by ELISA
OD.sub.450 nm Antibody concentration (nM) Clone ID 30 10 3.333
1.111 0.370 0.123 0.041 Blank 78B2D7 1.54 1.56 1.41 1.38 1.16 0.67
0.36 0.17 78A11F6 2.06 2.07 2.00 1.92 1.50 0.91 0.45 0.16 105C5D7
1.53 1.56 1.5 1.32 1.19 0.67 0.34 0.15 73D3G2 1.81 2.09 2.03 1.81
1.29 0.72 0.36 0.19 IgG control 0.12 0.15 0.11 0.16 0.12 0.11 0.11
0.13
TABLE-US-00009 TABLE 9 Binding activities of chimeric anti-PD-L1
mAbs to PD-L1 and PD-L2, as measured by ELISA OD.sub.450 nm Clone
ID hPD-L1 hPD-L2 78B2D7 2.708 0.218 78A11F6 2.7051 0.2053 105C5D7
2.636 0.214 73D3G2 2.749 0.156 IgG control 0.206 0.189
[0231] (Part B) Detection of the Binding of Anti-PD-L1 Antibodies
to Cells Expressing PD-L1 by Flow Cytometry
[0232] CHO-K1 cells were stably transfected with a pIRES plasmid
containing the nucleic acid sequence encoding full length human
PD-L1 (see Example 2, step 2) to generate CHO-K1 cells stably
expressing human PD-L1 (herein referred to as CHO-K1-hPD-L1 cells).
Other CHO-K1 were stably transfected with a pIRES plasmid
containing the nucleic acid sequence encoding full length cyno
PD-L1 to generate CHO-K1 cells stably expressing cyno PD-L1 (herein
referred to as CHO-K1-cPD-L1 cells). CHO-K1-hPD-L1 and
CHO-K1-cPD-L1 cells were cultured and expanded in T-75 culture
flasks to 90% confluence. The culture media was aspirated, and the
cells were washed twice with HBSS (Hanks Balanced Salt Solution,
Invitrogen). The cells were treated with enzyme-free cell
dissociation solution (Versene solution, Invitrogen) and collected.
The cells were then washed twice with HBSS, cell counts were
determined, and cells were resuspended with HBSS at
2.times.10.sup.6 cells/mL. Goat serum was added to the cell
suspension to a final concentration of 1%, and the cells were
blocked for 30 minutes on ice and then washed twice with HBSS. The
cells were collected after centrifugation and resuspended in FACS
buffer (HBSS+1% BSA, v/v) at 2.times.10.sup.6 cells/mL. 100 .mu.L
of the cell suspension were then added to each well of 96-well
plate. 100 .mu.L of purified anti-PD-L1 antibodies from Example 2
were added to each well of the 96-well plate and incubated for 2
hours on ice. Cells were washed twice with FACS buffer, and 100
.mu.L of Alexa 488-labeled secondary antibody (Invitrogen) were
added to the 96-well plate and incubated for 1 hour on ice. The
samples were washed three times with FACS buffer, and 100 .mu.L
fixation buffer (4% paraformaldehyde v/v) were added to each well
and incubated for 10 minutes. The cells were then washed twice with
FACS buffer and resuspended in 100 .mu.L FACS buffer. The mean
fluorescence intensity (MFI) was determined using FACS Calibur
(BD), and the results are shown in FIGS. 7-8 and in Tables 10-11.
The IgG control was human IgG, and the values in the tables are the
mean fluorescence intensity of the cell population.
TABLE-US-00010 TABLE 10 Binding activities of chimeric anti-PD-L1
mAbs to CHO-K1-hPD-L1, as determined by FACS Mean fluorescence
intensity Antibody concentration (nM) Clone ID 100.00 33.33 11.11
3.70 1.23 0.41 0.14 0.00 78B2D7 82.63 83.24 87.56 94.76 98.11 65.29
34.11 2.38 78A11F6 232.0 225.0 209.5 199.1 174.2 104.6 51.5 232.0
105C5D7 181.70 193.73 200.84 202.98 206.24 137.04 73.16 4.30 73D3G2
124.25 116.01 113.68 112.98 106.26 87.01 44.27 3.12 IgG control
100.00 33.33 11.11 3.70 1.23 0.41 0.14 0.00 Mean fluorescence
intensity Antibody concentration (nM) Clone ID 100.00 10 1 0.1 0.01
0.001 0.0001 0.00 73G11B3 214.65 212.97 106.85 17.21 3.96 1.88 1.87
2.14 127D2F3 234.35 235.41 88.55 15.54 5.31 2.10 4.04 3.82 192D9E7
146.45 133.63 97.58 23.50 7.85 2.66 3.58 3.45 IgG control 2.70 2.73
2.51 2.55 1.70 1.75 1.63 1.64
TABLE-US-00011 TABLE 11 Binding activities of chimeric anti-PD-L1
mAbs to CHO-K1-cPD-L1, as determined by FACS Mean fluorescence
intensity Antibody concentration (nM) Clone ID 100.00 33.33 11.11
3.70 1.23 0.41 0.14 0.00 78B2D7 96.17 91.80 100.83 108.37 115.06
71.57 37.56 2.94 78A11F6 144.0 143.0 141.8 138.7 124.0 74.5 38.3
144.0 105C5D7 120.27 133.00 135.86 141.93 136.76 83.81 41.97 2.81
73D3G2 137.47 136.81 136.19 126.29 116.22 83.18 44.31 3.75 IgG
control 3.33 2.94 2.93 2.73 2.82 2.69 2.74 2.57 Mean fluorescence
intensity Antibody concentration (nM) Clone ID 100.00 10 1 0.1 0.01
0.001 0.001 0.00 73G11B3 106.84 99.16 94.87 19.84 3.83 2.02 1.78
1.75 127D2F3 116.91 104.57 80.49 14.29 3.08 1.91 1.78 1.75 192D9E7
87.94 82.32 77.17 17.00 3.38 1.93 1.75 1.74 IgG control 8.11 1.95
1.83 2.20 1.87 1.90 7.33 1.73
[0233] (Part C) Binding Affinity and Dissociation Constant of
Anti-PD-L1 Antibodies
[0234] Dissociation constants were determined by Octed red 96
(Fortiebio). The detailed operation and methods were followed
according to the specifications of the instrument provided by the
manufacturer. Briefly, a streptavidin sensor (SA sensor, Fortiebio)
was used for the affinity determination. Biotinylated
PD-L1.sup.ECD-hFc (Immunogen A) was diluted to 10 .mu.g/mL in PBS
buffer (pH 7.4) containing 0 1% (w/w) BSA and 0.02% (v/v) Tween20
and incubated with the streptavidin sensor. Five different
concentrations of anti-PD-L1 antibody were incubated with the
Immunogen A-loaded streptavidin sensor at 30.degree. C. for 3
minutes. The reaction mixture was further incubated in PBS buffer
(pH 7.4) containing 0.1% (v/w) BSA and 0.02% (v/v) Tween20 at
30.degree. C. for 5 minutes. The association and dissociation
signals of anti-PD-L1 antibodies to Immunogen A were recorded in
real time using Octet Red 96. The affinity, association and
dissociation constants were determined using Octet User software,
and the results are shown in Table 12.
TABLE-US-00012 TABLE 12 Binding kinetics and affinities of chimeric
anti-PD-L1 mAbs to human PD-L1.sup.ECD-hFc protein, as determined
by Octet Red 96 Clone ID K.sub.D (nM) k.sub.a (1/Ms) k.sub.d (1/s)
78B2D7 1.37E-10 8.40E+05 1.15E-04 78A11F6 1.48E-10 2.45E+05
3.64E-05 105C5D7 2.45E-10 1.50E+06 3.68E-04 73D3G2 <1.0E-12
3.98E+05 <1.0E-07 73G11B3 1.16E-10 3.41E+05 3.95E-05 127D2F3
<1.0E-12 3.68E+05 <1.0E-07 192D9E7 5.19E-11 5.06E+05
2.63E-05
Example 4
Determination of the Ability of the Anti-PD-L1 Antibodies to Block
the Binding of PD-L1 to its Binding Partners PD-1 and B7.1
[0235] Protein-based receptor ligand binding assays were performed
to determine the ability of the anti-PD-L1 antibodies to block the
binding of PD-L1 to its binding partners PD-1 and B7.1.
[0236] Biotinylated recombinant PD-1.sup.ECD and B7.1.sup.ECD
proteins were prepared as described for biotinylated recombinant
PD-L1.sup.ECD-hFc in Example 2. The extracellular domain of PD-1
corresponds to amino acids Leu25-Glu167 of Uniprot database protein
Q15116, and the extracellular domain of B7.1 corresponds to amino
acids Val35 -Asn242 of Uniprot database protein NP_005182.
[0237] Purified PD-L1.sup.ECD-hFc (Example 2) was diluted with PBS
to a final concentration of 1.0 .mu.g/mL, and 100 .mu.L diluted
PD-L1.sup.ECD-hFc were added to each well of a 96-well plate that
was then sealed with plastic film and incubated at 4.degree. C.
overnight. The plate was washed twice with wash buffer (PBS+0.01%
(v/v) Tween 20) and incubated with blocking buffer (PBS+0.01% (v/v)
BSA+1% Tween 20 (w/w)) at room temperature for 2 hours. The
blocking buffer was aspirated, and 50 .mu.L purified anti-PD-L1
antibodies from Example 2 were added to each well of 96-well plate.
100 .mu.L of biotinylated recombinant PD-1.sup.ECD or B7.1.sup.ECD
protein were added to each well, mixed, and incubated at 37.degree.
C. for 2 hours. The plate was washed three times with wash buffer
(PBS+0.01% (v/v) Tween 20). 100 .mu.L HRP-conjugated streptavidin
(Sigma) were then added to each well and incubated at 37.degree. C.
for 2 hours. The plate was then washed three times with wash
buffer, and 100 .mu.L TMB substrate were added to each well. After
30 minutes of incubation at room temperature, the reaction was
stopped by adding 100 .mu.L stop solution (0.1N HCl). The
absorbance at OD450 nm was measured with an ELISA plate reader
(384plus SpectraMax, Molecular Devices). The results, shown in
FIGS. 9-10, demonstrate that the anti-PD-L1 antibodies can block
the binding of PD-L1 to its binding partners, PD-1 and B7.1.
Example 5
PBMC Stimulation Assay to Examine the Ability of the Anti-PD-L1
Antibodies to Block the Binding of PD-L1 to its Binding Partners
PD-1 and B7.1
[0238] (A) A T cell stimulation assay was performed to examine the
effect of the anti-PD-L1 antibodies on T cell stimulation by their
blocking of the binding of PD-L1 to its binding partners PD-1 and
B7.1.
[0239] (Step 1) Isolation of Peripheral Blood Mononuclear Cells
(PBMCs) from Whole Blood by a Ficoll Gradient.
[0240] Whole blood was diluted with PBS at a ratio of 1:1 (v/v) and
gently added on top of Ficoll solution (GE Healthcare) using a
sterile pipette. The volume ratio of Ficoll to diluted whole blood
was 3:4. The samples were centrifuged at 400 g at 20.degree. C. for
30 minutes. Three layers of solution were formed after
centrifugation, with the upper layer being plasma, and the middle
milk white layer being mononuclear cells. A sterile pipette was
used to collect the mononuclear cells from the middle layer and
transfer them to a new centrifugal tube. Three times the sample
volume of PBS was added, and the samples were centrifuged at 100 g
at room temperature for 10 minutes. The supernatant was discarded,
and the lymphocytes were resuspended with 10 mL PBS buffer. The
lymphocytes were washed with PBS three times to remove blood
platelets. The lymphocyte suspension was then resuspended with 10
mL RPMI 1640 culture medium (Invitrogen) containing 10% FBS.
[0241] (Step 2) PBMC Stimulation Test
[0242] Constructs encoding full-length PD-L1 protein were generated
by subcloning PD-L1 nucleotide sequence into pIRES plasmid
(pIRES-puro-PD-L1). To anchor anti-CD3 (OKT3) (see Kipriyanov et
al., 1997, Peds. 10:445-453) into the cell membrane, OKT3 scFv was
fused to the C-terminus of mouse CD8a (amino acids 113-220 of NCBI
accession No: NP--1074579.1) and subcloned into pIRES-OS8 (see
Sambrook and Russell, Id.). pIRES-puro-PD-L1 and pIRES-OS8 were
co-transfected to CHO-K1 and Hep3B cells following the preparation
method in Example 2 to generate the stable cell lines
CHO-K1-PD-L1/OS8 and Hep3B-PD-L1/OS8. The cells were used to
stimulate T lymphocytes. Before the experiments, CHO-K1-PD-L1/OS8
and Hep3B-PD-L1/OS8 cells were treated with 10 .mu.g/mL mitomycin
at 37.degree. C. for 3 hours.
[0243] 100 .mu.L PBMC (containing 5.times.10.sup.4 cells) were
added to the wells of a 96-well plate, and the test antibody
solution was then added to the 96-well plate and incubated for 15
minutes at room temperature. 50 .mu.L of 5.times.10.sup.3
CHO-K1-PD-L1/OS8 or Hep3B-PD-L1/OS8 were added to each well and
cultured at 37.degree. C., 5% CO.sub.2 for 72 hours. The
supernatants were collected and stored at -20.degree. C. until
analysis.
[0244] (Step 3) Detection of Interferon Gamma (IFN-.gamma.)
Secretion by ELISA
[0245] Quantification of the levels of IFN-.gamma. in culture
supernatant was carried out using Human IFN-gamma Quantikine ELISA
Kit (R&D Systems SIF50), following the manufacturer-provided
operating instructions and kit reagents. Briefly, the IFN-.gamma.
polyclonal antibodies were coated onto the ELISA microplates, and
400 .mu.L of culture supernatant as well as the standard were added
to each well and incubated at room temperature for 2 hours. The
plates were washed 4 times with wash buffer, followed by the
addition of HRP-conjugated anti-human IFN-.gamma. antibodies, and
incubated at room temperature for 2 hours. After washes, a
chromogenic substrate was added and incubated in the dark at room
temperature for 30 minutes, and the reaction was terminated by the
addition of a stop solution. The absorbance at 450 nm was
determined using an ELISA plate reader, and the results, shown in
FIG. 11-12 and in Table 13, demonstrate that the anti-PD-L1
antibodies analyzed by the PBMC lymphocyte stimulation test can
increase IFN-.gamma. secretion. The IgG control is human IgG, and
the values listed in the table are the IFN-.gamma. concentrations
in the culture supernatant (pg/mL).
TABLE-US-00013 TABLE 13 Chimeric anti-PD-L1 mAbs induce IFN-.gamma.
release from stimulated PBMCs IFN-.gamma. (pg/mL) Antibody
Concentration (.mu.g/mL) Clone ID 1 0.1 0.01 0.001 78B2D7 7706.15
7930 1760.5 2452.75 78A11F6 13030.6 6100.8 11073.4 3742.75 105C5D7
7570.65 5850.5 3381.6 1642.75 73D3G2 8760.05 9557.75 3280.1 3831.2
IgG Control 3147.2 1610.25 2482.75 2086.8 IFN-.gamma. (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 10 1 0.1 0.01 0.001
73G11B3 10684.16 7861.03 6291.91 5704.91 2404.27 127D2F3 10085.22
8096.69 7606.29 7028.66 3378.52 192D9E7 15661.38 8303.26 7465.29
501.27 602.06 IgG Control 1717.42 1134.51 442.03 315.84 1034.55
Example 6
Mixed Lymphocyte Reaction Assay to Examine the Ability of the
Anti-PD-L1 Antibodies to Block the Binding of PD-L1 to its Binding
Partners PD-1 and B7.1
[0246] (B) A mixed lymphocyte reaction was performed to examine the
impact of the anti-PD-L1 antibodies on T cell stimulation by their
blocking of the binding of PD-L1 to its binding partners PD-1 and
B7.1.
[0247] (Step 1) Isolation and Culture of Dendritic Cells from Human
CD14+ Cells
[0248] Ficoll Paque Plus (GE Healthcare) was used to isolate PBMCs
from whole blood, following the manufacturer-provided instructions.
The protocol was the same as that described in Example 5A, step
1.
[0249] PBMCs were resuspended in RPMI 1640 complete medium
containing 10% FBS, and the cell concentration was adjusted to
1.times.10.sup.5 cells/mL. The cells were cultured in T-75 culture
flasks at 37.degree. C., 5% (v/v) CO.sub.2 for 2 hours. The culture
supernatant and non-adhered cells were transferred to a new T-75
culture flask, and the original T-75 culture flask was replenished
with new RPMI 1640 complete culture medium supplemented with 10%
FBS and incubated at 37.degree. C. and 5% (v/v) CO.sub.2 for 2
hours. The culture supernatant and non-adhered cells were removed,
and the adherent cells were replenished with RPMI 1640 culture
media containing 10% FBS and incubated at 37.degree. C., 5% (v/v)
CO.sub.2 for 18 hours. The culture supernatant and non-adhered
cells were removed, and the adherent cells were replenished with
RPMI 1640 complete culture media supplemented with 500 U/mL
recombinant human GM-CSF (PEPROTECH) and 500 U/mL recombinant human
interleukin IL-4 (PEPROTECH) and cultured for 4 days. After 4 days,
the culture media was replenished with complete RPMI 1640 media
supplemented with GM-CSF and IL-4, and the cells were cultured for
an additional 2 days. The culture medium was then replaced with
RPMI 1640 complete media with 1 .mu.g/mL LPS and incubated for 18
hours. Dendritic cells were then collected by adding PBS containing
EDTA and centrifuging at 300 g for 5 minutes. The supernatant was
aspirated, and the cells were washed once more with PBS. The
collected human CD14+ dendritic cells were resuspended in RPMI 1640
complete media, and the cell counts were determined.
[0250] (Step 2) Isolation and Purification of Human CD4+ T
Cells.
[0251] Human CD4+ T cells were isolated and purified from PBMCs
using a MagCellect.TM. human CD4 +T Cell Isolation Kit (R&D
Systems), following the manufacturer-provided instructions.
[0252] (Step 3) Mixed Lymphocyte Reaction
[0253] Purified CD4+ cells from different healthy volunteers were
co-cultured with dendritic cells in 96-well plates. The cell
densities were adjusted to 10.sup.5 cells per 80 .mu.L. 10.sup.5
purified CD4+ T cells and 2.times.10.sup.4 dendritic cells were
added to each well of a 96-cell plate, purified anti-PD-L1
antibodies from Example 2 were added to the appropriate wells in
the plate, and the plates were incubated at 37.degree. C. in a 5%
CO.sub.2 incubator. The supernatants were collected at day 3 for
IL-2 measurement and at day 5 for IFN.gamma. measurement, and the
cytokine levels were determined.
[0254] (Step 4) Measurement of IFN-.gamma. and IL-2 Levels in
Supernatant by ELISA
[0255] Quantification of the levels of IFN-.gamma. or IL-2 in the
supernatants collected in the previous step was carried out using
Human IFN-gamma Quantikine ELISA Kit (R&D Systems SIF50) and
Human IL-2 Quantikine ELISA Kit D2050 (R&D Systems S2050),
respectively, following the manufacturer-provided operating
instructions and kit reagents.
[0256] The results for IL-2, shown in FIGS. 14, 21-22, 25-26 and in
Tables 14-19, show that the IL-2 level increased with an increase
in anti-PD-L1 antibody concentrations. The IgG control is human
IgG, and the values listed in Tables 14-19 are the IL-2
concentration (pg/mL) in the culture supernatants. PBMC donor 0, 1,
2, etc. refer to blood donor ID.
TABLE-US-00014 TABLE 14 Chimeric anti-PD-L1 mAbs induce IL-2
release, as determined by a mixed lymphocyte reaction assay (PBMC
donor 0) IL-2 (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 78B2D7 158.6 243.9 328.3 377.1 349.6 73D3G2
162.6 350.2 321.8 315.6 326.6 IgG Control 204.2 200.5 86.9 95.7
ND
TABLE-US-00015 TABLE 15 Chimeric anti-PD-L1 mAbs induce IL-2
release, as determined by a mixed lymphocyte reaction assay (PBMC
donor 2) IL-2 (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 78B2D7 67.9 46.8 98.3 122.1 136.0 73D3G2 56.5
81.7 99.5 106.9 115.0 IgG Control 46.6 42.2 59.7 51.6 ND
TABLE-US-00016 TABLE 16 Chimeric PD-L1 mAbs induce IL-2 release, as
determined by a mixed lymphocyte reaction assay (PBMC donor 1) IL-2
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
78A11F6 52.9 104.15 145.25 140.2 IgG Control 72.953 57.35 51.7
59.7
TABLE-US-00017 TABLE 17 Chimeric PD-L1 mAbs induce IL-2 release, as
determined by a mixed lymphocyte reaction assay (PBMC donor 2) IL-2
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
78A11F6 91.6 119.0 121.3 108.1 IgG Control 75.7 55.7 82.0 89.4
TABLE-US-00018 TABLE 18 Chimeric PD-L1 mAbs induce IL-2 release, as
determined by a mixed lymphocyte reaction assay (PBMC donor1) IL-2
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
10 73G11B3 215.74 364.97 453.08 426.22 476.65 127D2F3 226.15 273.92
428.72 496.61 440.14 192D9E7 211.11 370.99 473.09 461.01 487.74 IgG
Control 216.01 227.40 261.45 227.63 224.80
TABLE-US-00019 TABLE 19 Chimeric PD-L1 mAbs induce IL-2 release, as
determined by a mixed lymphocyte reaction assay (PBMC donor 2) IL-2
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
73G11B3 54.25 90.07 109.84 139.67 127D2F3 68.18 100.47 71.53 101.24
192D9E7 73.42 129.90 119.75 94.42 IgG Control 70.40 76.46 77.52
62.43
[0257] The results for IFN-.gamma., shown in FIGS. 13, 15-20, 23-24
and in Tables 20-27, show that the IFN-.gamma. level increased with
an increase in anti-PD-L1 antibody concentrations. The IgG control
is human IgG, and the values listed in Tables 19-26 are the
IFN-.gamma. concentration (pg/mL) in the culture supernatants.
TABLE-US-00020 TABLE 20 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 105C5D7 890.3 2059.3 5704.3 6895.8 11382.3
73D3G2 662.8 1303.8 1784.5 2824.2 1450.7 IgG Control 830.7 1048.9
1188.5 1184.2 1453.8
TABLE-US-00021 TABLE 21 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 105C5D7 1618.9 3742.0 11369.5 9793.0 18112.0
73D3G2 2601.9 2257.3 5937.2 2569.7 4377.8 IgG Control 3241.3 1291.2
2098.7 1813.2 3882.0
TABLE-US-00022 TABLE 22 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 78B2D7 1005 8089 14211 11455 IgG Control 2121 3449
4312 3661
TABLE-US-00023 TABLE 23 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 78B2D7 7360 15246 37996 27143 31872 IgG Control
3826 8030 4453 7078 ND
TABLE-US-00024 TABLE 24 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma.(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001
0.01 0.1 1 10 73G11B3 1524.46 3745.46 5742.85 6484.36 6947.89
127D2F3 599.23 1562.17 3010.79 2836.19 4836.78 192D9E7 220.86
1999.12 3123.87 3300.21 4075.74 IgG Control 1125.86 575.61 458.84
1060.62 1282.99
TABLE-US-00025 TABLE 25 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma.(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001
0.01 0.1 1 73G11B3 3340.70 3394.80 7476.55 16357.65 127D2F3 6036.85
6284.00 8764.25 12618.35 192D9E7 4731.43 8098.33 9064.80 12352.45
IgG Control 4165.90 4844.30 4544.18 5225.38
TABLE-US-00026 TABLE 26 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma.(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001
0.01 0.1 1 78A11F6 3676.55 6445.75 11702.6 9551.35 IgG Control
3970.28 3083.65 3709.35 4529.95
TABLE-US-00027 TABLE 27 Chimeric PD-L1 mAbs induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma.(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001
0.01 0.1 1 78A11F6 3840.7 5861.8 14758.55 23066.4 IgG Control
3634.94 3659.05 5517.55 5415.7
Example 7
Determination of the Amino Acid Sequences in the Variable Regions
of the Anti-PD-L1 Antibodies
[0258] Total RNA isolation: After the supernatants from the
hybridoma subclones obtained from Example 2 were characterized
(i.e., validation and determination of bioactivity, Examples 3-5),
5.times.10.sup.7 hybridoma cells were collected by centrifugation.
1 mL Trizol was added to the cell pellets, mixed and transferred to
1.5 mL centrifuge tubes, and incubated at room temperature for 5
minutes. 0.2 mL chloroform was added to the samples and vortexed
for 15 seconds. After standing for 2 minutes, the mixtures were
centrifuged at 12000 g at 4.degree. C. for 5 minutes. The
supernatants were collected and transferred to new 1.5 mL
centrifuge tubes, 0.5 mL isopropyl alcohol was added and mixed
gently, and the samples were incubated at room temperature for 10
minutes. The samples were centrifuged at 12000 g at 4.degree. C.
for 15 minutes. The supernatants were aspirated, and the
precipitates were washed with 1 mL 75% (v/v) ethanol. The mixtures
were centrifuged at 12000 g at 4.degree. C. for 5 minutes, the
supernatants were decanted, and the precipitates were air-dried.
Total RNA was obtained by adding DEPC-treated water to the
precipitates (55.degree. C. water bath for 10 minutes).
[0259] Reverse transcription and PCR: 1 .mu.g of RNA and reverse
transcriptase were added to a reaction mixture of a final volume of
20 .mu.L, and the mixture was incubated at 42.degree. C. for 60
minutes and then at 70.degree. C. for 10 minutes to terminate the
reaction. A 50 .mu.L PCR reaction mixture was prepared, containing
1 .mu.L cDNA, 25 pmol of each primer, 1 .mu.L DNA polymerase, 250
.mu.mol dNTPs, and the buffer system. The PCR program settings were
as follows: denaturation at 95.degree. C. for 3 minutes, 35 cycles
of denaturation (95.degree. C. for 30 seconds), annealing
(55.degree. C. for 30 seconds) and elongation (72.degree. C. for 35
seconds), followed by a final extension at 72.degree. C. for 5
minutes to obtain the PCR product. The commercially available
reverse transcription kit used was PrimeScript RT Master Mix
(Takara, RR036), and the commercially available Q5 ultra-fidelity
polymerase PCR kit was from NEB (M0492).
[0260] Cloning and sequencing: 5 .mu.L PCR products were examined
by agarose gel electrophoresis, and the samples were recovered from
the agarose gel using NucleoSpin Gel & PCR Clean-up kit
(MACHEREY-NAGEL, 740609). Ligation reaction: To a 50 ng sample, 50
ng T vector, 0.5 .mu.L ligase, and 1 .mu.L buffer were added and
brought to a final volume of 10 .mu.L with water. The reaction
mixture was incubated at 16.degree. C. for 30 minutes using the T4
DNA Ligase (NEB, M0402). 5 .mu.L ligation product was added to 100
.mu.L competent cells (Ecos 101 competent cells, Yeastern, FYE607),
which were incubated on ice for 5 minutes, heat shocked at
42.degree. C. for 1 minute and incubated on ice again for 1 minute.
Cells were recovered by adding 650 .mu.L SOC medium without
antibiotics and incubating at 37.degree. C. in a shaking incubator
for 30 minutes at 200 rpm. 200 .mu.L of each bacterial culture were
spread onto an LB agar plate containing antibiotics at 37.degree.
C. overnight. The next day, PCR reactions were set up using the T
vector primers M13F and M13R. Pipette tips were used to pick
bacterial colonies and were dipped into PCR reaction mixture and
pipetted up and down. Half of the reaction mixture was transferred
to an LB agar plate containing 100 nM ampicillin. After the PCR
reactions, 5 .mu.L of the PCR products were removed and examined by
agarose gel electrophoresis, and positive samples were sent for
sequencing and analysis (Kabat, 1991, "Sequences of proteins of
objective interest," the NIH, Bethesda, Md.). The sequencing
results are shown in Tables 1-2.
Example 8
Conversion, Expression and Purification of Fully Human Anti-PD-L1
Antibody
[0261] (step 1) Plasmid construction and preparation: Sequences of
the anti-PD-L1 antibody heavy and light chain variable regions were
obtained according to Example 6. The anti-PD-L1 antibodies' heavy
chain variable region sequences were subcloned into expression
vectors containing a signal peptide and a human heavy chain IgG4
constant region with a S228P mutation. The anti-PD-L1 antibodies'
light chain variable region sequences were subcloned into
expression vectors containing a signal peptide and a human antibody
light chain kappa constant region. The recombinant plasmids were
verified and confirmed by sequencing (the sequencing method was the
same as in Example 6) Alkaline lysis was performed using a reagent
kit (MACHEREY-NAGEL) to improve the purity and quality of the
recombinant plasmids, and the plasmids were filtered through 0.22
uM filters (Millpore). The purified plasmids were used for
transfection.
[0262] (step 2) Transfection: HEK293E cells (Invitrogen) were
cultured in FreeStyle 293 medium (Invitrogen) at 37.degree. C., 130
RPM, 8% CO.sub.2 (v/v). HEK293E cells were adjusted to
1-1.5.times.10.sup.6/mL cell density for transfection. 10% (v/v)
F68 (Invitrogen) was added to the FreeStyle 293 medium to a final
concentration of 0.1% (v/v), as Medium A. 5 mL of Medium A and 200
.mu.g/mL PEI (Sigma) were mixed to generate Medium B. 5 mL of
Medium A and 100 .mu.g/mL recombinant plasmid from step 1 were
mixed to generate Medium C. After 5 minutes of incubation, Medium B
and Medium C were mixed and incubated for 15 minutes to generate
Mixture D. 10 mL of Mixture D were slowly added to 100 mL HEK293E
cells with continuous stirring to avoid local accumulation of PEI.
HEK293E cells were incubated overnight while shaking The next day,
peptone was added to a final concentration of 0.5% (w/v). On about
day 5-7, the antibodies' titers were determined. On about day 6-7,
the HEK293E cultures were centrifuged (30 minutes, 3500 RPM), and
the supernatants were collected and filtered through 0.22 uM
filters for purification.
[0263] (step 3) Antibody purification: Protein A columns (GE) were
washed with 0.1M NaOH for 30 minutes or with 5 bed volumes of 0.5M
NaOH to get rid of endotoxin. Columns that had not been used in a
long time were soaked in 1M NaOH for at least 1 hour, washed with
endotoxin-free water to a neutral pH, and washed with 10 bed
volumes of 1% Triton X100. The columns were then equilibrated with
5 bed volumes of PBS (PBS phosphate buffer, pH 7.2). The filtered
supernatants from step 2 were loaded onto the columns, and the flow
through was collected, if necessary. The columns were washed with 5
bed volume of PBS and then eluted with 5 bed volumes of 0.1M
Glycine-HCl pH 3.0. The eluates containing anti-PD-L1 antibodies
were neutralized with 0.5 bed volumes of 1M Tris-HCl (NaCl 1.5M) pH
8.5. The human anti-PD-L1 antibodies were dialyzed in 1.times. PBS
for 4 hours, to avoid endotoxin contamination After dialysis, the
anti-PD-L1 antibody concentrations were determined by
spectrophotometry or a reagent kit, the purities of the antibodies
were determined by HPLC-SEC, and the contents of endotoxin were
determined by an endotoxin test kit (Lonza). The fully human
anti-PD-L1 antibodies were characterized, and the results are shown
in FIGS. 27-52, and in Tables 28-46. FIGS. 27-28 and Tables 28-29
show the binding of the fully human anti-PD-L1 antibodies to human
and cyno PD-L1-ECD by ELISA. FIGS. 29-30 and Tables 30-31 show the
binding of the fully human anti-PD-L1 antibodies to CHOK1 cells
expressing human and cyno PD-L1 by FACS. FIGS. 31-32 show that the
fully human anti-PD-L1 antibodies block the binding of PD-L1 to its
binding partners PD-1 and B7.1. FIGS. 33-42 and Tables 32-41 show
fully human anti-PD-L1 antibodies increase IFN-.gamma. and IL-2
release in a mixed lymphocyte reaction. FIGS. 43-47 and Tables
42-46 show fully human anti-PD-L1 antibodies increase IFN-.gamma.
and IL-2 release in a T cell stimulation assay.
TABLE-US-00028 TABLE 28 Binding activities of fully human
anti-PD-L1 mAbs to human PD-L1.sup.ECD-hFc, as measured by ELISA
OD.sub.450 nm Antibody concentration (nM) Clone ID 100 10 1 0.1
0.01 0.001 0.0001 0 78A11F6 2.8201 2.8900 2.8894 1.9965 0.3824
0.1756 0.1577 0.1530 73D3G2 2.8966 3.0116 2.9859 2.1088 0.3933
0.1772 0.1523 0.1481 IgG control 0.7673 0.2250 0.1571 0.1492 0.1538
0.1494 0.1617 0.1476 OD.sub.450 nm Antibody concentration (nM)
Clone ID 30 3 0.3 0.03 0.003 0.0003 0.00003 0 73G11B3 2.6663 2.7190
2.4782 0.8546 0.4034 0.2541 0.2564 0.2808 127D2F3 2.5856 2.6826
2.4835 0.8524 0.3293 0.2639 0.2806 0.2933 192D9E7 2.5863 2.6732
2.5213 0.7856 0.3220 0.2850 0.2866 0.3043 IgG control 0.3154 0.2771
0.2935 0.2726 0.2982 0.3375 0.2958 0.3094
TABLE-US-00029 TABLE 29 Binding activities of fully human
anti-PD-L1 mAbs to cyno PD-L1.sup.ECD-hFc, as measured by ELISA
OD.sub.450 nm Antibody concentration (nM) Clone ID 100 10 1 0.1
0.01 0.001 0.0001 0 78A11F6 2.5798 2.5706 2.5334 1.1198 0.2264
0.1193 0.1025 0.1047 73D3G2 2.6203 2.68412 2.5759 1.0655 0.2196
0.1110 0.1011 0.1008 IgG control 0.3983 0.1336 0.0989 0.0967 0.0987
0.0972 0.0988 0.1003 OD.sub.450 nm Antibody concentration (nM)
Clone ID 30 3 0.3 0.03 0.003 0.0003 0.00003 0 73G11B3 2.6000 2.6474
2.5191 0.9997 0.3582 0.2827 0.3076 0.3283 127D2F3 2.5911 2.5976
2.4895 0.8892 0.3323 0.2803 0.2975 0.3063 192D9E7 2.5373 2.6620
2.5503 0.9525 0.3761 0.3482 0.3244 0.3868 IgG control 0.3064 0.2823
0.2803 0.2764 0.3022 0.2730 0.3098 0.3237
TABLE-US-00030 TABLE 30 Binding activities of fully human
anti-PD-L1 mAbs to CHOK1-hPD-L1 as measured by FACS Mean
fluorescence intensity Antibody concentration (nM) Clone ID 300 30
3 0.3 0.03 0.003 0.0003 0 78B2D7 251.14 283.60 339.31 117.20 22.94
8.78 7.85 5.00 78A11F6 632.85 619.94 618.98 217.30 36.51 10.37 6.53
5.41 105C5D7 381.81 380.05 434.68 116.25 23.07 7.30 6.61 4.49
73D3G2 601.77 595.07 575.90 192.63 28.61 7.60 4.68 4.25 IgG control
4.86 5.73 3.56 3.16 2.92 3.03 2.79 2.80
TABLE-US-00031 TABLE 31 Binding activities of fully human
anti-PD-L1 mAbs to CHO-K1-cPD-L1, as determined by FACS Mean
fluorescence intensity Antibody concentration (nM) Clone ID 300 30
3 0.3 0.03 0.003 0.0003 0 78B2D7 258.06 302.42 326.16 108.44 46.38
32.68 5.48 5.56 78A11F6 537.74 529.33 521.27 243.47 64.08 8.94 5.47
5.02 105C5D7 298.58 335.16 374.56 106.56 18.78 9.85 5.31 4.62
73D3G2 512.04 518.93 495.31 173.64 26.17 7.84 4.87 4.57 IgG control
15.57 4.12 3.91 3.73 3.79 3.62 3.76 3.64
TABLE-US-00032 TABLE 32 Fully human PD-L1 mAbs induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 78B2D7
53.1 56.5 137.5 164.8 105C5D7 43.0 92.2 118.2 148.3 73D3G2 59.7
83.4 113.9 139.0 IgG Control 46.6 42.2 59.7 51.6
TABLE-US-00033 TABLE 33 Fully human PD-L1 mAb induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 78A11F6
84.25 118.5 101 134 IgG Control 72.953 57.35 51.7 59.7
TABLE-US-00034 TABLE 34 Fully human PD-L1 mAb induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 10
78A11F6 89.58 114.61 154.78 224.87 245.20 192D9E7 64.11 86.42
137.19 204.73 189.68 IgG Control 63.64 74.67 67.98 73.29 67.97
TABLE-US-00035 TABLE 35 Fully human PD-L1 mAb induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 10
73G11B3 573.57 648.95 868.96 1014.24 1064.57 127D2F3 544.22 665.75
848.72 933.23 1023.55 IgG Control 446.43 458.51 435.26 445.36
426.00
TABLE-US-00036 TABLE 36 Fully human PD-L1 mAb induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 10
78A11F6 210.20 282.54 729.50 735.75 841.73 73D3G2 205.47 217.89
563.30 716.45 791.34 73G11B3 215.00 274.06 653.58 728.96 601.30
192D9F7 210.13 340.53 712.86 721.05 769.60 IgG Control 160.49
177.03 212.03 160.97 82.83
TABLE-US-00037 TABLE 37 Fully human PD-L1 mAb induce IL-2 release,
as determined by a mixed lymphocyte reaction assay IL-2 (pg/mL)
Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1 105C5D7
42.96 92.19 118.21 148.25 IgG Control 46.58 42.19 59.70 51.57
TABLE-US-00038 TABLE 38 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 78A11F6 1080.31 1651.88 3005.76 3762.96 3397.09
105C5D7 1882.71 2582.91 4933.23 4280.52 5263.90 73D3G2 956.82
1079.33 1992.68 3039.33 2593.15 192D9E7 592.35 915.35 1874.37
2763.14 2273.32 IgG Control 608.70 1003.94 1126.40 727.47
636.27
TABLE-US-00039 TABLE 39 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 73G11B3 2058.01 2267.26 4172.51 4129.22 5808.68
127D2F3 1475.48 2458.15 4090.74 4724.47 7296.25 IgG Control 931.67
571.68 514.44 740.05 570.71
TABLE-US-00040 TABLE 40 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 78A11F6 3135.38 4290.55 7534.50 9726.43 9124.71
IgG Control 4007.92 3836.12 3300.70 4397.55 3242.08
TABLE-US-00041 TABLE 41 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a mixed lymphocyte reaction assay
IFN-.gamma. (pg/mL) Antibody Concentration (.mu.g/mL) Clone ID
0.001 0.01 0.1 1 10 73D3G2 12543.45 10023.02 20961.33 21464.90
21374.43 73G11B3 9788.65 10670.98 21742.25 21670.28 16944.51
192D9E7 9030.83 11220.04 19646.05 22885.80 15520.20 IgG Control
6111.26 6615.51 7673.03 7619.53 8977.26
TABLE-US-00042 TABLE 42 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a T cell stimulation assay IFN-.gamma.
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
10 78A11F6 822.73 1384.48 3269.42 3452.41 2322.47 73D3G2 855.85
1446.70 3278.93 3712.56 2646.34 IgG Control 806.00 758.23 1058.48
998.00 807.39
TABLE-US-00043 TABLE 43 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a T cell stimulation assay IFN-.gamma.
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
10 105C5D7 7366.89 13819.46 19665.04 22400.88 21581.53 IgG Control
6648.67 8381.33 5917.55 8390.70 8109.32
TABLE-US-00044 TABLE 44 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a T cell stimulation assay IFN-.gamma.
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
73G11B3 6780.00 10874.50 13064.00 13520.00 127D2F3 5085.50 11225.00
9345.00 14071.00 192D9F7 6152.50 13119.00 11531.50 15173.50 IgG
Control 6087.50 5106.50 6746.50 7074.50
TABLE-US-00045 TABLE 45 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a T cell stimulation assay IFN-.gamma.
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
10 78A11F6 4027.63 9195.29 9838.49 9963.87 12499.25 73D3G2 4446.93
10640.60 12326.57 12281.83 13962.35 73G11B3 5122.60 6337.46 9360.13
8791.74 13065.92 IgG Control 3173.89 3002.36 3349.96 2843.70
3170.99
TABLE-US-00046 TABLE 46 Fully human PD-L1 mAb induce IFN-.gamma.
release, as determined by a T cell stimulation assay IFN-.gamma.
(pg/mL) Antibody Concentration (.mu.g/mL) Clone ID 0.001 0.01 0.1 1
127D2F3 5447.74 13802.78 28155.63 46846.94 192D9E7 6829.27 13482.85
15450.80 12798.11 IgG Control 5043.61 4999.09 7599.12 7754.80
Example 9
Determination of Binding and Dissociation Constants by Biacore
[0264] Anti-human Fc IgG was immobilized on flow cells 1 and 2:
HBS-EP+ (10 mM HEPES, 150 mM NaCl, 3 mM EDTA, 0.05% P20, pH 7.4)
was used as miming buffer, and the immobilization of anti-human Fc
IgG was carried out using the immobilization wizard template. Flow
cells 1 and 2 of a Series S CM5 sensor chip were activated with
freshly-mixed 50 mmol/L NHS and 200 mmol/L EDC. 20 .mu.g/mL of
anti-human Fc IgG diluted in 10 mM NaAC (pH 4.5) was injected into
the activated flow cells 1 and 2. The remaining active coupling
sites were blocked with 1M ethanolamine.
[0265] Recombinant His-tagged hPD-L1 ECD protein was diluted to 50
nM, followed by four 2-fold serial dilutions with HBS-EP+ buffer.
The His-tagged hPD-L1 ECD protein concentrations were 0 nM, 3.125
nM, 6.25 nM, 12.5 nM, 25 nM and 50 nM. K.sub.D measurements were
carried out with HBS-EP+ as the running buffer. Each antibody was
injected over the CM5 sensor flow cell 2 with a flow rate of 10
.mu.L/min to reach response 230 RU. Prepared His-tagged hPD-L1 ECD
protein was then injected over flow cells 1 and 2, at a flow rate
of 30 .mu.L/min for 180 sec. Buffer flow was maintained for 400
seconds for dissociation measurements (30 .mu.L/min). To remove the
tested antibody from the surface, 10 mM glycine-HCl pH 1.5 was
injected for 20 seconds (30 .mu.L/min). Flow cell 1 was used as
reference flow cell. The above steps were repeated for each
concentration of serially-diluted His-tagged PD-L1.sup.ECD protein.
The K.sub.D value for each antibody was evaluated using Biacore
T200 evaluation software 1.0, and the data was fit with a 1:1
binding model. The results are shown in Table 47.
TABLE-US-00047 TABLE 47 Binding kinetics and affinities of fully
human anti-PD-L1 mAbs to His-tagged human PD-L1 ECD protein, as
determined by Biacore Clone ID K.sub.D (M) k.sub.a (1/Ms) k.sub.d
(1/s) 78A11F6 6.430E-10 7.336E+5 4.717E-4 73D3G2 5.358E-10 1.414E+6
7.573E-4
Example 10
ADCC and CDC Effector Function Analysis
[0266] To confirm the presumed absence of effector function of
fully human anti-PD-L1 antibodies, antibody-dependent cell-mediated
cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)
assays were performed.
[0267] For the ADCC assay, samples of PD-L1-expressing HCC827 cells
were adjusted to a concentration of 12.5.times.10.sup.4 cells/mL
with ADCC medium (without phenol red). 40 .mu.L of cell suspensions
(1.times.10.sup.4 viable cells) were added to each well of a
v-bottom 96-well plate. 20 .mu.L of antibody were serially diluted
in ADCC medium (without phenol red) and added to each well in
triplicate. The plate was incubated at 22-25.degree. C. for 30
minutes. NK92 cells stably transfected with Fc.gamma.R III 158V
were adjusted with ADCC medium (without phenol red) so that by
adding 40 .mu.L of NK92 cells stably transfected with Fc.gamma.R
III 158V to the target cells, the ratio of effector to target cells
was 1:1. The plate was then incubated at 37.degree. C. for 4 hours.
After 4 hours incubation, 100 .mu.L substrate from CytoTox 96 kit
(Promega) were added to each well. For maximum cell lysis control,
2 .mu.L of lysis solution (CytoTox-ONE.TM. kit, Promega) was added
10 minutes before the addition of 100 .mu.L substrate from CytoTox
96 kit. The plate was incubated at room temperature for 10 minutes,
and 50 .mu.L of stop solution were then added to each well and
mixed for 30 seconds. The absorbance at 560/590 nm was measured,
and the 1% lysis values were calculated using GraphPad Prism 5.0.
The results, shown in FIG. 48, show that the positive control
anti-EGFR antibody induced ADCC in HCC827 cells, but the fully
human anti-PD-L1 antibodies had no ADCC effect on HCC827 cells.
[0268] For the CDC assay, HCC827 cells were adjusted to
1.25.times.10.sup.6 cells/mL with cell culture medium. To a
flat-bottom 96-well white plate, 40 .mu.L/well of cells were added.
40 .mu.L/well of fully human anti-PD-L1 antibody, serially diluted
in CDC medium, were added to each well in duplicate. The plates
were incubated in the hood for 30 minutes. Commercially available
purified human complement (Quidel, cat #042637) was added to the
plates containing cells at 20 .mu.L/well to a final concentration
of 20%. The plates were incubated at 37.degree. C. for 20 hours.
The Celltiter-glo luminescent cell viability kit (Promega, No.
G7573) was used to test cell viability according to the
manufacture-provided protocol. The plates were shaken on a
microplate shaker for 2 minutes at a speed of 200 and then
incubated at room temperature for 10 minutes. The luminescence
signal was read with Envision. For data analysis, the %
cytotoxicity was calculated using GraphPad Prism 5.0. The results,
shown in FIG. 49, show that the positive control anti-EGFR antibody
induced CDC in HCC827 cells, but the fully human anti-PD-L1
antibodies had no CDC effect on HCC827 cells.
Example 11
Antibody Thermostability, Measured by Differential Scanning
Calorimetry (DSC)
[0269] Fully human anti-PD-L1 antibodies were adjusted to 1 mg/mL
and a final volume of about 700 .mu.L with sample buffer. The
parameters were set up as follows (VP-DSC): Starting Temperature
30.degree. C.; Final Temperature 100.degree. C.; Scan rate
50.degree. C./hour; Number of Rescans 0; PreScan Thermostat 3 min;
PostScan Thermostat 0 min; Post Cycle Thermostat 25.degree. C.;
Filtering Period 25 seconds; Feedback Mode/Gain None; Cell Refill
Parameters 35.degree. C. The resulting protein-buffer thermograms
were processed by subtracting a corresponding buffer-buffer scan
and subsequently fitting a baseline to the trace. The Tms were
recorded at each peak maxima observed in the thermograms using
Origin.TM. 7.0 software. The results are shown in FIG. 50.
Example 12
Antibody Freeze/Thaw Stability
[0270] The freeze/thaw stabilities of the fully human anti-PD-L1
antibodies were characterized as follows. A 100 .mu.L aliquot from
the frozen stocks of each anti-PD-L1 antibody was thawed at room
temperature. Once fully thawed, the samples were then rapidly
frozen in the -80.degree. C. freezer and kept at -80.degree. C. for
at least two hours before being thawed again at room temperature.
The samples went through three identical freeze/thaw cycles. Visual
inspection was used to check for precipitation. 20 .mu.L aliquots
were removed from the samples for size-exclusion chromatography
(SEC) analysis after three freeze/thaw cycles. The stability of the
fully human anti-PD-L1 antibodies before and after the freeze/thaw
cycles were analyzed by HPLC-SEC characterization. The results,
shown in FIG. 51, demonstrated that after three freeze/thaw cycles,
monomer IgGs accounted for more than 95% of each of the anti-PD-L1
antibodies tested.
Example 13
Antibody Solubility
[0271] The solubility of the fully human anti-PD-L1 antibodies was
characterized by concentrating 10 mg IgG using centrifugal filters
(Amicon Ultra--0.5 mL 30K) at 14000 g at 4.degree. C. down to
>100 mg/mL. 2 mL or more of IgG was added into the centrifugal
filters and concentrated at 14000 g at 4.degree. C. The setting
time of centrifuging was 2 min, 3 min, 5 min, 8 min, 15 min, and 20
min, and each time 20 .mu.L were aliquoted to a collection tube to
measure the concentration with a nanodrop at A280. The
centrifugation was finished when the concentration reached 100
mg/mL. For HPLC-SEC characterization, 6 .mu.L of concentrated
samples were injected into an HPLC-SEC column, and the percentages
of monomers and aggregates were determined based on the peak area.
The results, shown in FIG. 52, demonstrated that all of the fully
human anti-PD-L1 antibodies tested had a solubility over 100 mg/mL
and that monomer IgGs were higher than 95% for all of the
anti-PD-L1 antibodies tested.
[0272] While the invention has been described in detail, and with
reference to specific embodiments thereof, it will be apparent to
one of ordinary skill in the art that various changes and
modifications can be made therein without departing from the spirit
and scope of the invention.
REFERENCES
[0273] Blank et al., 2005, Cancer Immunotherapy, 54:307-314 [0274]
Bowers N L, Helton E S, Huijbregts R P H, et al. PLoS Pathog. 2014
March; 10 (3): e100399 [0275] Dong H, Zhu Tamada K, Chen L. Nature
Med. 1999; 5:1365-1369 [0276] Freeman et al., 2000, J. Exp. Med.,
192:1027-34 [0277] Harding and Lonberg, 1995, Ann. N.Y. Acad. Sci.
764:536-546 [0278] Hirano et al., 2005, Cancer Res., 65:1089-96
[0279] Kabat, 1991, "Sequences of proteins of objective interest,"
the NIH, Bethesda, Md. [0280] Kipriyanov et al., 1997, Peds.
10:445-453 [0281] Lonberg and Huszar, 1995, Internal Rev. Immunol.
13:65-93 [0282] Lonberg et al., 1994, Nature 368: 856-859 [0283]
Sambrook and Russell, 1989, Molecular cloning: a laboratory manual,
New York: Cold Spring Harbor Laboratory Press, 2nd ed. [0284] Sheng
Yao 2013 Nat Rev Drug Discov. 2013 12(2): 130-146 [0285] Weinstock
and McDermott, 2015, Ther Adv Urol., 7(6):365-77 [0286] Wherry,
2011, Nat. Immunol., 12:492-99 [0287] Zippelius et al., 2015,
Cancer Immunol Res.; 3(3):236-44 [0288] Zou and Chen L, 2008, Nat
Rev Immunol., 8(6):467-77
Sequence CWU 1
1
781120PRTHomo sapiens 1Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Arg Leu Trp Phe Gly Gly Val Met Asp Ala Trp Gly Gln
100 105 110 Gly Ala Ser Val Thr Val Ser Ser 115 120 211PRTHomo
sapiens 2Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser 1 5 10
318PRTHomo sapiens 3Ala Asp Ile Lys Gln Asp Gly Ser Glu Lys Tyr Tyr
Val Asp Ser Val 1 5 10 15 Lys Gly 411PRTHomo sapiens 4Asp Arg Leu
Trp Phe Gly Gly Val Met Asp Ala 1 5 10 5107PRTHomo sapiens 5Asp Ile
Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15
Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20
25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu
Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg
Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile
Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln
Gln Tyr Asn Ser Tyr Ser Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys
Val Glu Ile Lys 100 105 611PRTHomo sapiens 6Arg Ala Ser Gln Ser Ile
Ser Ser Trp Leu Ala 1 5 10 77PRTHomo sapiens 7Lys Ala Ser Ser Leu
Glu Ser 1 5 89PRTHomo sapiens 8Gln Gln Tyr Asn Ser Tyr Ser Trp Thr
1 5 9121PRTHomo sapiens 9Glu Val Gln Leu Val Glu Ser Gly Gly Gly
Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala
Ser Gly Ile Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg
Gln Thr Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys
Gln Asp Gly Ser Glu Lys Tyr Tyr Val Lys Ser Val 50 55 60 Lys Gly
Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80
Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85
90 95 Val Arg Asp Arg Ala Val Pro Gly Thr Gly Ala Phe Asp Ile Trp
Gly 100 105 110 Gln Gly Thr Met Val Ile Val Ser Ser 115 120
1010PRTHomo sapiens 10Gly Ile Thr Phe Ser Ser Tyr Trp Met Ser 1 5
10 1117PRTHomo sapiens 11Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Lys Ser Val Lys 1 5 10 15 Gly 1212PRTHomo sapiens 12Asp Arg
Ala Val Pro Gly Thr Gly Ala Phe Asp Ile 1 5 10 13107PRTHomo sapiens
13Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn
Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr
Cys Gln Gln Tyr Asp Ser Ser Leu Phe 85 90 95 Thr Phe Gly Gln Gly
Thr Lys Leu Glu Ile Lys 100 105 1411PRTHomo sapiens 14Arg Ala Ser
Gln Ser Ile Ser Asn Trp Leu Ala 1 5 10 157PRTHomo sapiens 15Lys Ala
Ser Ser Leu Glu Ser 1 5 169PRTHomo sapiens 16Gln Gln Tyr Asp Ser
Ser Leu Phe Thr 1 5 17119PRTHomo sapiens 17Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met
Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45
Ala Asn Ile Lys Gln Asp Gly Asn Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val
Tyr Tyr Cys 85 90 95 Ala Arg Asn Ile Lys Trp Gly Asp Ala Phe Asp
Ile Trp Gly Gln Gly 100 105 110 Thr Met Val Thr Val Ser Ser 115
1811PRTHomo sapiens 18Ser Gly Phe Thr Phe Ser Ser Tyr Trp Met Ser 1
5 10 1918PRTHomo sapiens 19Ala Asn Ile Lys Gln Asp Gly Asn Glu Lys
Tyr Tyr Val Asp Ser Val 1 5 10 15 Lys Gly 2010PRTHomo sapiens 20Asn
Ile Lys Trp Gly Asp Ala Phe Asp Ile 1 5 10 21107PRTHomo sapiens
21Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1
5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Ser
Tyr 20 25 30 Leu Asn Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys
Leu Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro
Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu
Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Ile Tyr Ser
Cys Gln Gln Ser Tyr Ser Ile Pro Leu 85 90 95 Thr Phe Gly Gly Gly
Thr Lys Val Glu Ile Lys 100 105 2211PRTHomo sapiens 22Arg Ala Ser
Gln Ser Ile Ser Ser Tyr Leu Asn 1 5 10 237PRTHomo sapiens 23Ala Ala
Ser Ser Leu Gln Ser 1 5 249PRTHomo sapiens 24Gln Gln Ser Tyr Ser
Ile Pro Leu Thr 1 5 25121PRTHomo sapiens 25Glu Val Gln Leu Val Glu
Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu
Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Thr Tyr 20 25 30 Trp Met
Asn Trp Val Arg Gln Ala Pro Gly Arg Gly Leu Glu Trp Val 35 40 45
Ala Asn Ile Lys Gln Asp Gly Gly Glu Lys Tyr Tyr Val Asp Ser Val 50
55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu
Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Met
Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Arg Ser Trp Tyr Pro Asp Ala
Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Val Val Thr Val Ser Ser
115 120 2611PRTHomo sapiens 26Ser Gly Phe Thr Phe Asn Thr Tyr Trp
Met Asn 1 5 10 2718PRTHomo sapiens 27Ala Asn Ile Lys Gln Asp Gly
Gly Glu Lys Tyr Tyr Val Asp Ser Val 1 5 10 15 Lys Gly 2812PRTHomo
sapiens 28Asp Gly Arg Ser Trp Tyr Pro Asp Ala Phe Asp Ile 1 5 10
29106PRTHomo sapiens 29Asp Ile Gln Met Thr Gln Ser Pro Ser Thr Leu
Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala
Ser Gln Ser Ile Ser Thr Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys
Leu Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Glu Val Ser Ile
Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser
Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Asp
Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr His Ser Phe Ser Ser 85 90
95 Phe Gly Gly Gly Thr Arg Val Glu Ile Lys 100 105 3011PRTHomo
sapiens 30Arg Ala Ser Gln Ser Ile Ser Thr Trp Leu Ala 1 5 10
317PRTHomo sapiens 31Glu Val Ser Ile Leu Glu Ser 1 5 328PRTHomo
sapiens 32Gln Gln Tyr His Ser Phe Ser Ser 1 5 33121PRTHomo sapiens
33Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1
5 10 15 Ser Leu Arg Leu Ser Cys Val Val Ser Gly Phe Thr Phe Ser Ser
Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln Thr Pro Gly Lys Gly Leu
Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln Asp Gly Ser Glu Lys Tyr
Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg Leu Thr Ile Ser Arg Asp
Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg
Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Val Arg Asp Arg Ala
Val Pro Gly Thr Gly Ala Leu Asp Ile Trp Gly 100 105 110 Gln Gly Thr
Met Val Thr Val Ser Ser 115 120 347PRTHomo sapiens 34Gly Phe Thr
Phe Ser Ser Tyr 1 5 356PRTHomo sapiens 35Lys Gln Asp Gly Ser Glu 1
5 3612PRTHomo sapiens 36Asp Arg Ala Val Pro Gly Thr Gly Ala Leu Asp
Ile 1 5 10 37107PRTHomo sapiens 37Asp Ile Gln Met Thr Gln Ser Pro
Ser Thr Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr
Cys Arg Ala Ser Gln Ser Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr
Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Lys
Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60
Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65
70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr
Leu Phe 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100
105 3811PRTHomo sapiens 38Arg Ala Ser Gln Ser Ile Ser Ser Trp Leu
Ala 1 5 10 397PRTHomo sapiens 39Lys Ala Ser Ser Leu Glu Ser 1 5
409PRTHomo sapiens 40Gln Gln Tyr Asn Ser Tyr Leu Phe Thr 1 5
41119PRTHomo sapiens 41Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asn Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Leu Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Lys Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Ala Arg Asp Pro Ile Tyr Ser Ser Tyr Phe Asp Tyr Trp Gly Gln Gly
100 105 110 Ile Leu Val Thr Val Ser Ser 115 427PRTHomo sapiens
42Gly Phe Thr Phe Ser Ser Tyr 1 5 436PRTHomo sapiens 43Lys Gln Asp
Gly Ser Glu 1 5 4410PRTHomo sapiens 44Asp Pro Ile Tyr Ser Ser Tyr
Phe Asp Tyr 1 5 10 45107PRTHomo sapiens 45Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Asn Tyr 20 25 30 Leu Asn
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Phe Ala Ala Tyr Tyr Cys Gln Gln Ser Tyr Gly
Ile Pro Phe 85 90 95 Thr Phe Gly Pro Gly Thr Lys Val Asp Ile Lys
100 105 4611PRTHomo sapiens 46Arg Ala Ser Gln Ser Ile Ser Asn Tyr
Leu Asn 1 5 10 477PRTHomo sapiens 47Ala Ala Ser Ser Leu Gln Ser 1 5
489PRTHomo sapiens 48Gln Gln Ser Tyr Gly Ile Pro Phe Thr 1 5
49120PRTHomo sapiens 49Glu Val Gln Leu Val Glu Ser Gly Gly Gly Leu
Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser
Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Trp Met Ser Trp Val Arg Gln
Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Asp Ile Lys Gln
Asp Gly Ser Glu Lys Tyr Tyr Val Asp Ser Val 50 55 60 Lys Gly Arg
Phe Thr Ile Ser Arg Asp Asn Ala Lys Asn Ser Leu Tyr 65 70 75 80 Leu
Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90
95 Thr Arg Asp Lys Pro His Trp Gly Gly Val Met Asp Ala Trp Gly Gln
100 105 110 Gly Thr Ser Val Thr Val Ser Ser 115 120 507PRTHomo
sapiens 50Gly Phe Thr Phe Ser Ser Tyr 1 5 516PRTHomo sapiens 51Lys
Gln Asp Gly Ser Glu 1 5 5211PRTHomo sapiens 52Asp Lys Pro His Trp
Gly Gly Val Met Asp Ala 1 5 10 53107PRTHomo sapiens 53Asp Ile Gln
Met Thr Gln Ser Pro Ser Thr Leu Ser Ala Ser Glu Gly 1 5 10 15 Asp
Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Ser Ile Ser Arg Trp 20 25
30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile
35 40 45 Tyr Lys Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe
Ser Gly 50 55 60 Ser Gly Ser Gly Thr Glu Phe Thr Leu Thr Ile Ser
Ser Leu Gln Pro 65 70 75 80 Asp Asp Phe Ala Thr Tyr Tyr Cys Gln Gln
Tyr His Ser Tyr Ser Trp 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val
Glu Ile Lys 100 105 5411PRTHomo sapiens 54Arg Ala Ser Gln Ser Ile
Ser Arg Trp Leu Ala 1 5 10 557PRTHomo sapiens 55Lys Ala Ser Ser Leu
Glu Ser 1 5 569PRTHomo sapiens 56Gln Gln Tyr His Ser Tyr Ser Trp
Thr 1 5 57360DNAHomo sapiens 57gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt
agctattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtggccgac ataaagcaag atggaagtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtgc
gagagatcga 300ctatggttcg ggggggttat ggatgcctgg ggtcaaggag
cttcagtcac tgtctcctca 36058363DNAHomo sapiens 58gaggtgcagc
tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag
cctctggaat cacctttagt agctattgga tgagctgggt ccgccaaact
120ccagggaagg ggctggagtg ggtggccaac ataaagcaag atggaagtga
gaaatactat 180gtaaagtctg tgaagggccg attcaccatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgt gagagatcga 300gcagtgcctg gtacgggggc
ttttgatatc tggggccaag ggacaatggt catcgtctct 360tca 36359357DNAHomo
sapiens 59gaggtgcagc tggtggagtc tgggggaggc ttggtccagc
ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt agctattgga
tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg ggtggccaac
ataaagcaag atggaaatga gaaatactat 180gtggactctg tgaagggccg
attcaccatc tccagagaca acgccaagaa ctcactgtat 240ctgcaaatga
acagcctgag agccgaggac acggctgtat attactgtgc gagaaatatt
300aagtggggag atgcttttga tatctggggc caagggacaa tggtcaccgt ctcttca
35760363DNAHomo sapiens 60gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttaat
acctattgga tgaactgggt ccgccaggct 120ccagggaggg ggctggagtg
ggtggccaac ataaaacaag atggaggtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ctacaaatga acagcctgag agccgaggac acggctatgt attactgtgc
gagagatggc 300cgcagctggt accctgatgc ttttgatatc tggggccaag
ggacagtggt caccgtctct 360tca 36361321DNAHomo sapiens 61gacatccaga
tgacccagtc tccttccacc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggccagtca gagtattagt agctggttgg cctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacag
tataatagtt attcgtggac gttcggccaa 300gggaccaagg tggaaatcaa a
32162321DNAHomo sapiens 62gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
aactggttgg cctggtatca gcagaaacca 120gggaaagccc ctaagctcct
gatctataag gcgtctagtt tagaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct
240gatgattttg caacttatta ctgccaacag tatgatagtt ctttgttcac
ttttggccag 300gggaccaagc tggagatcaa a 32163321DNAHomo sapiens
63gacatccaga tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc
60atcacttgcc gggcaagtca gagcattagc agctatttaa attggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
ggtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg caatttactc ctgtcaacag
agttacagta tcccgctcac tttcggcgga 300gggaccaagg tggagatcaa a
32164318DNAHomo sapiens 64gacatccaga tgacccagtc tccttccacc
ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc gggccagtca gagtattagt
acctggttgg cctggtatca gcagaaactg 120gggaaagccc ctaaactcct
gatctatgag gtgtctattt tagaaagtgg ggtcccatca 180aggttcagcg
gcagtggatc tgggacagaa ttcactctca ccatcagcag cctgcagcct
240gatgattttg caacttatta ctgccaacag tatcatagtt tttcttcttt
cggcggaggg 300accagggtgg agatcaaa 31865363DNAHomo sapiens
65gaggtgcagc tggtggagtc tgggggaggc ttggtccagc ctggggggtc cctgagactc
60tcctgtgtag tctctggatt cacctttagt agctattgga tgagctgggt ccgccaaact
120ccagggaagg ggctggagtg ggtggccaat ataaagcaag atggaagtga
gaaatactat 180gtggactctg tgaagggccg actcaccatc tccagagaca
acgccaagaa ctcactgtat 240ctgcaaatga acagcctgag agccgaggac
acggctgtgt attactgtgt gagagatcga 300gcagtgcctg gaacgggggc
tttagatatc tggggccaag ggacaatggt caccgtctct 360tca 36366321DNAHomo
sapiens 66gacatccaga tgacccagtc tccttccacc ctgtctgcat ctgtaggaga
cagagtcacc 60atcacttgcc gggccagtca gagtattagt agctggttgg cctggtatca
gcagaaacca 120gggaaagccc ctaagctcct gatctataag gcgtctagtt
tagaaagtgg ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa
ttcactctca ccatcagcag cctgcagcct 240gatgattttg caacttatta
ctgccaacag tataatagtt atttgtttac ttttggccag 300gggaccaagc
tggagatcaa a 32167357DNAHomo sapiens 67gaggtgcagc tggtggagtc
tgggggaggc ttggtccagc ctggggggtc actgagactc 60tcctgtgtag cctctggatt
cacctttagt agctattgga tgagctgggt ccgccaggct 120ccagggaagg
ggctggagtg ggtggccaac ataaagcaag atggaagtga gaagtactat
180gtggactctg tgaagggccg attgaccatc tccagagaca acgccaagaa
ttcactgtat 240ctgcaaatga agagtctgag agccgaggac acggctgtgt
attactgtgc gagagatccc 300atatatagca gttactttga ctactggggc
cagggaatcc tggtcaccgt ctcctca 35768321DNAHomo sapiens 68gacatccaga
tgacccagtc tccatcctcc ctgtctgcat ctgtaggaga cagagtcacc 60atcacttgcc
gggcaagtca gagcattagc aactatttaa attggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctatgct gcatccagtt tgcaaagtgg
agtcccatca 180aggttcagtg gcagtggatc tgggacagat ttcactctca
ccatcagcag tctgcaacct 240gaagattttg cagcttacta ctgtcaacag
agttacggta tcccattcac tttcggccct 300gggaccaaag tggatatcaa a
32169360DNAHomo sapiens 69gaggtgcagc tggtggagtc tgggggaggc
ttggtccagc ctggggggtc cctgagactc 60tcctgtgcag cctctggatt cacctttagt
agctattgga tgagctgggt ccgccaggct 120ccagggaagg ggctggagtg
ggtggccgac ataaagcaag atggaagtga gaaatactat 180gtggactctg
tgaagggccg attcaccatc tccagagaca acgccaagaa ctcactgtat
240ctgcaaatga acagcctgag agccgaggac acggctgtgt attactgtac
gagagataag 300ccccactggg gaggggttat ggatgcctgg ggtcaaggaa
cttcagtcac tgtctcctca 36070321DNAHomo sapiens 70gacatccaga
tgacccagtc tccttccacc ctgtctgcat ctgaaggaga cagagtcacc 60atcacttgcc
gggccagtca gagtattagt aggtggttgg cctggtatca gcagaaacca
120gggaaagccc ctaagctcct gatctataag gcgtctagtt tagaaagtgg
ggtcccatca 180aggttcagcg gcagtggatc tgggacagaa ttcactctca
ccatcagcag cctgcagcct 240gatgattttg caacttatta ctgccaacaa
tatcatagtt attcgtggac gttcggccaa 300gggaccaagg tggaaatcaa a
32171221PRTHomo sapiens 71Phe Thr Val Thr Val Pro Lys Asp Leu Tyr
Val Val Glu Tyr Gly Ser 1 5 10 15 Asn Met Thr Ile Glu Cys Lys Phe
Pro Val Glu Lys Gln Leu Asp Leu 20 25 30 Ala Ala Leu Ile Val Tyr
Trp Glu Met Glu Asp Lys Asn Ile Ile Gln 35 40 45 Phe Val His Gly
Glu Glu Asp Leu Lys Val Gln His Ser Ser Tyr Arg 50 55 60 Gln Arg
Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn Ala Ala 65 70 75 80
Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr Arg Cys 85
90 95 Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile Thr Val Lys
Val 100 105 110 Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg Ile Leu Val
Val Asp Pro 115 120 125 Val Thr Ser Glu His Glu Leu Thr Cys Gln Ala
Glu Gly Tyr Pro Lys 130 135 140 Ala Glu Val Ile Trp Thr Ser Ser Asp
His Gln Val Leu Ser Gly Lys 145 150 155 160 Thr Thr Thr Thr Asn Ser
Lys Arg Glu Glu Lys Leu Phe Asn Val Thr 165 170 175 Ser Thr Leu Arg
Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr Cys Thr 180 185 190 Phe Arg
Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu Val Ile 195 200 205
Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr 210 215 220
72290PRTHomo sapiens 72Met Arg Ile Phe Ala Val Phe Ile Phe Met Thr
Tyr Trp His Leu Leu 1 5 10 15 Asn Ala Phe Thr Val Thr Val Pro Lys
Asp Leu Tyr Val Val Glu Tyr 20 25 30 Gly Ser Asn Met Thr Ile Glu
Cys Lys Phe Pro Val Glu Lys Gln Leu 35 40 45 Asp Leu Ala Ala Leu
Ile Val Tyr Trp Glu Met Glu Asp Lys Asn Ile 50 55 60 Ile Gln Phe
Val His Gly Glu Glu Asp Leu Lys Val Gln His Ser Ser 65 70 75 80 Tyr
Arg Gln Arg Ala Arg Leu Leu Lys Asp Gln Leu Ser Leu Gly Asn 85 90
95 Ala Ala Leu Gln Ile Thr Asp Val Lys Leu Gln Asp Ala Gly Val Tyr
100 105 110 Arg Cys Met Ile Ser Tyr Gly Gly Ala Asp Tyr Lys Arg Ile
Thr Val 115 120 125 Lys Val Asn Ala Pro Tyr Asn Lys Ile Asn Gln Arg
Ile Leu Val Val 130 135 140 Asp Pro Val Thr Ser Glu His Glu Leu Thr
Cys Gln Ala Glu Gly Tyr 145 150 155 160 Pro Lys Ala Glu Val Ile Trp
Thr Ser Ser Asp His Gln Val Leu Ser 165 170 175 Gly Lys Thr Thr Thr
Thr Asn Ser Lys Arg Glu Glu Lys Leu Phe Asn 180 185 190 Val Thr Ser
Thr Leu Arg Ile Asn Thr Thr Thr Asn Glu Ile Phe Tyr 195 200 205 Cys
Thr Phe Arg Arg Leu Asp Pro Glu Glu Asn His Thr Ala Glu Leu 210 215
220 Val Ile Pro Glu Leu Pro Leu Ala His Pro Pro Asn Glu Arg Thr His
225 230 235 240 Leu Val Ile Leu Gly Ala Ile Leu Leu Cys Leu Gly Val
Ala Leu Thr 245 250 255 Phe Ile Phe Arg Leu Arg Lys Gly Arg Met Met
Asp Val Lys Lys Cys 260 265 270 Gly Ile Gln Asp Thr Asn Ser Lys Lys
Gln Ser Asp Thr His Leu Glu 275 280 285 Glu Thr 290 73873DNAHomo
sapiens 73atgaggatat ttgctgtctt tatattcatg acctactggc atttgctgaa
cgcatttact 60gtcacggttc ccaaggacct atatgtggta gagtatggta gcaatatgac
aattgaatgc 120aaattcccag tagaaaaaca attagacctg gctgcactaa
ttgtctattg ggaaatggag 180gataagaaca ttattcaatt tgtgcatgga
gaggaagacc tgaaggttca gcatagtagc 240tacagacaga gggcccggct
gttgaaggac cagctctccc tgggaaatgc tgcacttcag 300atcacagatg
tgaaattgca ggatgcaggg gtgtaccgct gcatgatcag ctatggtggt
360gccgactaca agcgaattac tgtgaaagtc aatgccccat acaacaaaat
caaccaaaga 420attttggttg tggatccagt cacctctgaa catgaactga
catgtcaggc tgagggctac 480cccaaggccg aagtcatctg gacaagcagt
gaccatcaag tcctgagtgg taagaccacc 540accaccaatt ccaagagaga
ggagaagctt ttcaatgtga ccagcacact gagaatcaac 600acaacaacta
atgagatttt ctactgcact tttaggagat tagatcctga ggaaaaccat
660acagctgaat tggtcatccc agaactacct ctggcacatc ctccaaatga
aaggactcac 720ttggtaattc tgggagccat cttattatgc cttggtgtag
cactgacatt catcttccgt 780ttaagaaaag ggagaatgat ggatgtgaaa
aaatgtggca tccaagatac aaactcaaag 840aagcaaagtg atacacattt
ggaggagacg taa 87374984DNAArtificial Sequencehuman IgG4 constant
region with a S228P mutation 74gctagcacca agggcccttc cgtgttccct
ctggcccctt gctcccggtc cacctccgag 60tccaccgccg ctctgggctg tctggtgaag
gactacttcc ctgagcctgt gaccgtgagc 120tggaactctg gcgccctgac
ctccggcgtg cacaccttcc ctgccgtgct gcagtcctcc 180ggcctgtact
ccctgtcctc cgtggtgacc gtgccttcct cctccctggg caccaagacc
240tacacctgca acgtggacca caagccttcc aacaccaagg tggacaagcg
ggtggagtcc 300aagtacggcc ctccttgccc tccctgccct gcccctgagt
tcctgggcgg accctccgtg 360ttcctgttcc ctcctaagcc taaggacacc
ctgatgatct cccggacccc tgaggtgacc 420tgcgtggtgg tggacgtgtc
ccaggaagat cctgaggtcc agttcaattg gtacgtggat 480ggcgtggagg
tgcacaacgc caagaccaag cctcgggagg aacagttcaa ctccacctac
540cgggtggtgt ctgtgctgac cgtgctgcac caggactggc tgaacggcaa
ggaatacaag 600tgcaaggtca gcaacaaggg cctgccctcc tccatcgaga
aaaccatctc caaggccaag 660ggccagcctc gcgagcctca ggtgtacacc
ctgcctccta gccaggaaga gatgaccaag 720aatcaggtgt ccctgacatg
cctggtgaag ggcttctacc cttccgatat cgccgtggag 780tgggagagca
acggccagcc agagaacaac tacaagacca cccctcctgt gctggactcc
840gacggctcct tcttcctgta ctccaggctg accgtggaca agtcccggtg
gcaggaaggc 900aacgtctttt cctgctccgt gatgcacgag gccctgcaca
accactacac ccagaagtcc 960ctgtccctgt ctctgggcaa gtga
98475327PRTArtificial Sequencehuman IgG4 constant region with a
S228P mutation 75Ala Ser Thr Lys Gly Pro Ser Val Phe Pro Leu Ala
Pro Cys Ser Arg 1 5 10 15 Ser Thr Ser Glu Ser Thr Ala Ala Leu Gly
Cys Leu Val Lys Asp Tyr 20 25 30 Phe Pro Glu Pro Val Thr Val Ser
Trp Asn Ser Gly Ala Leu Thr Ser 35 40 45 Gly Val His Thr Phe Pro
Ala Val Leu Gln Ser Ser Gly Leu Tyr Ser 50 55 60 Leu Ser Ser Val
Val Thr Val Pro Ser Ser Ser Leu Gly Thr Lys Thr 65 70 75 80 Tyr Thr
Cys Asn Val Asp His Lys Pro Ser Asn Thr Lys Val Asp Lys 85 90 95
Arg Val Glu Ser Lys Tyr Gly Pro Pro Cys Pro Pro Cys Pro Ala Pro 100
105 110 Glu Phe Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro
Lys 115 120 125 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys
Val Val Val 130 135 140 Asp Val Ser Gln Glu Asp Pro Glu Val Gln Phe
Asn Trp Tyr Val Asp 145 150 155 160 Gly Val Glu Val His Asn Ala Lys
Thr Lys Pro Arg Glu Glu Gln Phe 165 170 175 Asn Ser Thr Tyr Arg Val
Val Ser Val Leu Thr Val Leu His Gln Asp 180 185 190 Trp Leu Asn Gly
Lys Glu Tyr Lys Cys Lys Val Ser Asn Lys Gly Leu 195 200 205 Pro Ser
Ser Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 210 215 220
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Gln Glu Glu Met Thr Lys 225
230 235 240 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro
Ser Asp 245 250 255 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu
Asn Asn Tyr Lys 260 265 270 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly
Ser Phe Phe Leu Tyr Ser 275 280 285 Arg Leu Thr Val Asp Lys Ser Arg
Trp Gln Glu Gly Asn Val Phe Ser 290 295 300 Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser 305 310 315 320 Leu Ser Leu
Ser Leu Gly Lys 325 76324DNAArtificial Sequencehuman antibody light
chain kappa constant region 76cgtacggtgg ctgcaccatc tgtcttcatc
ttcccgccat ctgatgagca gttgaaatct 60ggaactgcct ctgttgtgtg cctgctgaat
aacttctatc ccagagaggc caaagtacag 120tggaaggtgg ataacgccct
ccaatcgggt aactcccagg agagtgtcac agagcaggac 180agcaaggaca
gcacctacag cctcagcagc accctgacgc tgagcaaagc agactacgag
240aaacacaaag tctacgcctg cgaagtcacc catcagggcc tgagctcgcc
cgtcacaaag 300agcttcaaca ggggagagtg ttga 32477107PRTArtificial
Sequencehuman antibody light chain kappa constant region 77Arg Thr
Val Ala Ala Pro Ser Val Phe Ile Phe Pro Pro Ser Asp Glu 1 5 10 15
Gln Leu Lys Ser Gly Thr Ala Ser Val Val Cys Leu Leu Asn Asn Phe 20
25 30 Tyr Pro Arg Glu Ala Lys Val Gln Trp Lys Val Asp Asn Ala Leu
Gln 35 40 45 Ser Gly Asn Ser Gln Glu Ser Val Thr Glu Gln Asp Ser
Lys Asp Ser 50 55 60 Thr Tyr Ser Leu Ser Ser Thr Leu Thr Leu Ser
Lys Ala Asp Tyr Glu 65 70 75 80 Lys His Lys Val Tyr Ala Cys Glu Val
Thr His Gln Gly Leu Ser Ser 85 90 95 Pro Val Thr Lys Ser Phe Asn
Arg Gly Glu Cys 100 105 78663DNAHomo sapiens 78tttactgtca
cggttcccaa ggacctatat gtggtagagt atggtagcaa tatgacaatt 60gaatgcaaat
tcccagtaga aaaacaatta gacctggctg cactaattgt ctattgggaa
120atggaggata agaacattat tcaatttgtg catggagagg aagacctgaa
ggttcagcat 180agtagctaca gacagagggc ccggctgttg aaggaccagc
tctccctggg aaatgctgca 240cttcagatca cagatgtgaa attgcaggat
gcaggggtgt accgctgcat gatcagctat 300ggtggtgccg actacaagcg
aattactgtg aaagtcaatg ccccatacaa caaaatcaac 360caaagaattt
tggttgtgga tccagtcacc tctgaacatg aactgacatg tcaggctgag
420ggctacccca aggccgaagt catctggaca agcagtgacc atcaagtcct
gagtggtaag 480accaccacca ccaattccaa gagagaggag aagcttttca
atgtgaccag cacactgaga 540atcaacacaa caactaatga gattttctac
tgcactttta ggagattaga tcctgaggaa 600aaccatacag ctgaattggt
catcccagaa ctacctctgg cacatcctcc aaatgaaagg 660act 663
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